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Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate
Theriogenology 57 (2002) 1347±1355
Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate A.N. Fatehia,*, E.C. Zeinstraa, R.V. Kooijb, B. Colenbrandera, M.M. Beversa a
Department of Farm Animal Health, Utrecht University, Utrecht, The Netherlands Centre for Reproductive Medicine, IVF-laboratory, Neusserstr 111, 40489 DuÈsseldorf, Germany
b
Received 7 June 2001; accepted 27 July 2001
Abstract The aim of this study is to identify the effect of cumulus cells removal prior to the in vitro fertilization of matured bovine oocytes on cleavage rate. Denuded, matured oocytes were fertilized in presence or absence of loose cumulus cells, cumulus cell conditioned IVF medium (CCCM), charcoal-treated CCCM and charcoal-treated CCCM supplemented with progesterone at a ®nal concentration of 150 ng/ml. After 18 h of incubation with sperm, the presumptive embryos were cultured on a BRL monolayer and the percentage of cleaved embryos was evaluated on Day 4. Removal of cumulus cells prior to IVF signi®cantly reduced the cleavage rate (25% for denuded oocytes versus 56% for cumulus±oocyte complexes (COCs)). The addition of loose cumulus cells partially restored the effect of denudation (cleavage rate: 37% for denuded oocytes supplemented with loose cumulus cells versus 27% for denuded oocytes and 58% for COCs). CCCM also had a positive effect on the cleavage rate of oocytes denuded prior to IVF (36% for denuded oocytes fertilized in CCCM versus 14% for denuded oocytes). Treating the CCCM with charcoal resulted in complete loss of its effect on cleavage rate (18% for denuded oocytes fertilized in charcoal-treated CCCM versus 34% for denuded oocytes fertilized in CCCM). The addition of progesterone to charcoal-treated CCCM partially restored the reduction of the cleavage rate caused by charcoal treatment (27% for denuded oocytes fertilized in charcoal-treated CCCM supplemented with progesterone versus 14% for denuded oocytes fertilized in charcoal-treated CCCM and 36% for denuded oocytes fertilized in CCCM). In conclusion, removal of cumulus cells prior to IVF adversely affects the cleavage rate through loss of a factor secreted by these cells. This factor probably is progesterone. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Cumulus cells; Progesterone; IVF; COC * Corresponding author. E-mail address: [email protected] (A.N. Fatehi).
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 1 7 - 8
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1. Introduction The importance of cumulus cells for the maturation and acquisition of developmental competence in oocytes has been well-established [1±5]. Removal of cumulus cells from the oocytes prior to in vitro maturation adversely affects the maturation, fertilization and embryo development in rat [6], sheep [7] and cattle [5,8]. In almost all species a certain proportion of cumulus cells are still present at the site of fertilization [9,10]. In vitro fertilization in the presence of cumulus cells increased the fertilization rate in mouse [11±13], hamster [14], pig [15] and buffalo [16]. The mechanism by which cumulus cells facilitate fertilization is not clear. It has been suggested that cumulus cells are involved in the interaction between male and female gametes, guiding spermatozoa towards the oocytes [17,18], inducing capacitation [19,20], acrosome reaction [21,22], maintaining sperm motility and viability [22,23], preventing zona pellucida from precocious hardening [24±26] and enhancing sperm penetration and in vitro fertilization [19,22,27,28]. In cattle, Behalova and Greve [29], Cox [30] and Ball et al. [31] showed that the presence of cumulus cells surrounding the oocytes is not necessary for successful IVF. They reported almost equal rates of sperm penetration during IVF of cumulus-free and cumulus-enclosed oocytes. Hawk et al. [32] observed a negative effect of cumulus cells on sperm penetration, while Younis and Brackett [4], Cox et al. [19], and Zhang et al. [5] reported a positive effect of cumulus cells on the in vitro fertilization of bovine oocytes. The aim of this study is to investigate if and how the cumulus cells affect the in vitro fertilization of bovine oocytes. 2. Materials and methods 2.1. Collection of oocytes Bovine ovaries were collected at a slaughterhouse in a thermo¯ask and transported to the laboratory within 1 h. Excised ovaries were rinsed in physiological saline (0.9% NaCl) containing antibiotics (100 IU penicillin and 100 mg streptomycin per ml) and dried with paper towel. Cumulus±oocyte complexes (COCs) were aspirated from small antral follicles (2±8 mm diameter) using an 18 gauage needle attached to a tube in line with a vacuum pump. The COCs were selected on the presence of a multilayered compact cumulus investment and homogeneous ooplasm and randomly assigned to the various treatments. 2.2. In vitro maturation, fertilization and embryo culture In vitro maturation and IVF as well as in vitro embryo culture (IVC) took place at 39 8C in a humidi®ed atmosphere of 5% CO2 in air. Selected COCs were rinsed in HEPES buffered M199, (Gibco BRL, Paisely, UK) supplemented with 10% fetal calf serum (FCS). Groups of 35 were randomly allocated to each well of a 4 well culture plate (Nunc A/S, Roskilde, Denmark) containing 500 ml of maturation medium per well. They were then cultured for 23 h. Unless otherwise indicated the maturation medium consisted of tissue
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culture medium M199 (Gibco, Life Technologies, Breda, The Netherlands) supplemented with 10% FCS and 0.05 units/ml recombinant human FSH (Organon, Oss, The Netherlands). Frozen/thawed spermatozoa used for IVF were centrifuged over a percoll gradient for 30 min at 700 g at 25 8C. The sperm sample was collected by removing the gradient for the last 150 ml containing the sperm pellet. Thirty-®ve COCs or denuded oocytes were transferred to 430 ml of fertilization medium (Fert-TALP) as described by Parrish et al. [33] without glucose and 10 mg/ml penicillin/streptomycin instead of gentamycin. We added to each well 20 ml of sperm suspension (®nal concentration 0.5106 spermatozoa/ml), 20 ml heparin (®nal concentration 10 mg/ml) and 20 ml PHE (consisting of 20 mM D-penicillamin, 10 mM hypotaurine, and 1 mM epinephrine). After 18±20 h of incubation, the presumptive zygotes were freed from cumulus cells by vortexing for 3 min and the groups were randomly placed in a co-culture system of 0.5 ml M199 supplemented with 10% FCS on a monolayer of buffalo rat liver (BRL) cells in each well of a 4 well culture plate. On the fourth day of culture, the proportion of cleaved embryos was assessed. 2.3. BRL cell culture Buffalo rat liver (BRL) cells separated from the BRL cell line from the American Type Culture Collection (ATCC) were routinely cultured in a 1:1 mixture of Ham's F12 medium and Dulbecco's modi®ed Eagle's medium (Gibco) supplemented with 7.5% FCS (Gibco) and antibiotics. 2.4. Preparation of IVF conditioned medium A total of 23 h after incubation of 140 COCs in maturation medium, the attached cumulus cells were removed by vortexing for 3 min. The detached cumulus cells were recovered, then divided in to each well of a 4 well culture plate containing 500 ml of IVF medium. The suspension was incubated at 39 8C in 5% CO2 for 18 h. After incubation, the supernatant was ®ltered through a 0.22 mm ®lter (Millipore, Bedford, MA, USA). The ®ltrate was designated as cumulus cell conditioned medium (CCCM) and was used the same day as preparation. 2.5. Charcoal treatment of conditioned medium To remove small molecules including steroids [34], part of the CCCM was stirred for 10 min with 50 mg charcoal/ml (Norit: activated and neutralized; Sigma St. Louis, MO, USA) and then centrifuged at 4500 g for 10 min at room temperature. The supernatant was centrifuged again at 11; 600 g for 3 min and subsequently ®ltered through a 0.22 mm ®lter (Millipore) to remove charcoal particles. The ®ltered supernatant was designated as charcoal-treated CCCM. 2.6. Preparation of progesterone solution A stock solution of progesterone was freshly prepared by dissolving 0.15 mg progesterone (Sigma) in 1 ml DMSO. Using the stock solution, a solution of charcoal-treated
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CCCM supplemented with progesterone at a ®nal concentration of 150 ng/ml was prepared. The ®nal concentration of DMSO in this medium was 0.1% (v/v). 2.7. Experiments A series of ®ve experiments were carried out. Unless otherwise indicated IVM, IVF, and IVC were performed according to the procedures above. 2.7.1. Experiment 1 COCs were in vitro matured and the cumulus cells were subsequently removed. Denuded oocytes were in vitro fertilized and after IVF, the presumptive zygotes were cultured as described. Cultures in which COCs were stripped after IVF served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of nine replicates. 2.7.2. Experiment 2 COCs were in vitro matured and the cumulus cells were subsequently stripped. Denuded oocytes were in vitro fertilized in the presence or absence of the detached cumulus cells. Cultures in which COCs were stripped after IVF served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of three replicates. 2.7.3. Experiment 3 COCs were in vitro matured and the cumulus cells were subsequently stripped. Denuded oocytes were in vitro fertilized in CCCM. Denuded oocytes fertilized in plain IVF medium served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of four replicates. 2.7.4. Experiment 4 COCs were in vitro matured and the cumulus cells were subsequently stripped. Denuded oocytes were in vitro fertilized in charcoal-treated CCCM. Denuded oocytes fertilized in plain IVF medium served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of ®ve replicates. 2.7.5. Experiment 5 COCs were in vitro matured and the cumulus cells were subsequently stripped. Denuded oocytes were in vitro fertilized in charcoal-treated CCCM supplemented with progesterone. Denuded oocytes, fertilized in CCCM or charcoal-treated CCCM served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of ®ve replicates. 2.8. Statistical analysis The results were analyzed by Chi-square test and P < 0:05 was considered signi®cant.
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3. Results The removal of cumulus cells before in vitro fertilization of in vitro matured oocytes signi®cantly decreased the cleavage rate. (Table 1). When cumulus cells were removed by a narrow-bore pasteur pipette, instead of vortexing, a similar decrease of the cleavage rate was observed (data not shown). Addition of removed cumulus cell to the IVF medium of denuded oocytes partially restored the denudation effect and increased the cleavage rate (Table 2). In vitro fertilization of denuded oocytes in CCCM increased the cleavage rate (Table 3) as compared to the cleavage rate of denuded oocytes routinely in vitro fertilized in IVF medium. Treatment of CCCM with charcoal, resulted in a complete disappearance of its promoting effect on the cleavage rate of denuded oocytes following fertilization (Table 4). Table 1 The effect of cumulus cell removal before in vitro fertilization of in vitro matured bovine oocytes on cleavage rate
Cumulus-enclosed oocytes Cumulus-free oocytes
Oocytes (n)
Cleavage rate Day 4 (%)
1222 1092
684 (56) a 270 (25) b
a and b: P < 0:0001. Table 2 The effect of loose cumulus cells on the in vitro fertilization of denuded bovine oocytes
Cumulus-enclosed oocytes Denuded oocytes Denuded oocytes loose cumulus cells
Oocytes (n)
Cleavage rate Day 4 (%)
263 201 262
155 (58) a 54 (27) b 98 (37) c
a,b and a,c: P < 0:0001; b,c: P < 0:05. Table 3 The effect of cumulus cell conditioned medium (CCCM) on the cleavage rate of bovine oocytes denuded prior to IVF
Denuded oocytes Denuded oocytes CCCM
Oocytes (n)
Cleavage rate Day 4 (%)
640 479
95 (14) a 171 (36) b
a and b: P < 0:0001. Table 4 The effect of charcoal treatment of cumulus cell conditioned medium (CCCM) on the cleavage rate of bovine oocytes, denuded prior to IVF
Denuded oocytes Denuded oocytes CCCM Denuded oocytes charcoal-treated CCCM a and b: P < 0:0001.
Oocytes (n)
Cleavage rate Day 4 (%)
533 565 580
102 (19) a 193 (34) b 105 (18) a
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Table 5 The effect of charcoal-treated cumulus cell conditioned medium (CCCM) supplemented with progesterone on the cleavage rate of bovine oocytes denuded prior to IVF Denuded oocytes fertilized in
Oocytes (n)
Cleavage rate Day 4 (%)
CCCM Charcoal-treated CCCM Charcoal-treated CCCM progesterone
476 459 509
172 (36) a 66 (14) b 139 (27) c
a,b and b,c: P < 0:0001, a,c: P < 0:05.
Adding progesterone (150 ng/ml) to charcoal-treated CCCM partially restored the lost effect and resulted in a higher cleavage rate of bovine oocytes denuded prior to IVF (Table 5). 4. Discussion The results demonstrate that the presence of cumulus cells has a positive in¯uence on the in vitro fertilization of bovine oocytes. The cleavage rate signi®cantly decreases when cumulus cells are removed prior to in vitro fertilization. These results are in agreement with previous reports showing that the presence of cumulus cells facilitates fertilization of cattle oocytes leading to a higher penetration rate [19,21,35], higher cleavage rate [4,5,22,36] and a higher yield of blastocysts [5,36]. In other studies [29,30,31], it was found that removal of cumulus cells did not affect the penetration rate. However, male and female pronucleus formation was impaired [31] and a higher incidence of polyspermia was observed [29]. Cox et al. [19] reported that the positive effect of cumulus cells on fertilization is expressed only when the cells are in contact with the zona pellucida. In contrast, we found that the presence of loose cumulus cells partially restored the effect of denudation prior to in vitro fertilization. This suggests that both the presence of cumulus cells and cell±oocyte contact contribute to a proper fertilization process. The partial restoration of the fertilization rate might be due to the fact that the mechanical procedure of cumulus cell removal has harmed part of the oocytes, resulting in lower male pronuclear formation as suggested by Ball et al. [31]. The increase of cleavage rate of denuded oocytes by CCCM suggests that the positive effect of cumulus cells is the result of compound(s) secreted by cumulus cells. The fact that charcoal treatment inactivates the bene®cial effect of CCCM indicates that the secreted factor is a small molecule [33]. Progesterone probably is the active molecule since the addition of progesterone to charcoal-treated CCCM increased the fertilization rate of denuded oocytes. The lower fertilization rate of charcoal-treated CCCM supplemented with progesterone as compared to nontreated CCCM might be a concentration effect or may suggest that apart from progesterone, another small molecule is involved. Evidence that progesterone is the active molecule secreted by cumulus cells is supported by the study of Li et al. [37] which shows that COCs from 2 to 6 mm follicles produce progesterone. In our study, loose cumulus cells and CCCM almost equally increased the cleavage rate of denuded oocytes, suggesting that the amount of secreted progesterone was not signi®cantly
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different. Li et al. [37] observed a higher progesterone production by oocytectomized cumulus±oocyte complexes in the presence of IGF and FSH. As CCCM is prepared by the incubation of cumulus cells in routine fertilization medium, thus without IGF and FSH, the production of progesterone probably was not enhanced due to the absence of oocytes. A number of reports indicate that progesterone affects various sperm functions. Progesterone stimulates hyperactivity in human sperm [38] and increases the acrosome reaction in human [38], dog [39] and stallion sperm [40]. Moreover, progesterone in vitro enhances the number of stallion spermatozoa that binds to the zona pellucida [40]. In the case of dog [13] and stallion sperm [40], it has been shown that the effect of progesterone is exerted through a progesterone receptor localized in the plasma membrane of spermatozoa. Hence, it can be assumed that the positive effect of cumulus cells on the in vitro fertilization of bovine oocytes is at least partially caused by progesterone secreted by the cumulus cells. In conclusion, this study suggests that removal of cumulus cells from in vitro matured oocytes, prior to in vitro fertilization adversely affect the cleavage rate of cattle oocytes by the elimination of progesterone secreted by the cumulus cells.
Acknowledgements Grant sponsor: EU Commission; Grant number QLK3-CT1999-CT00104. The authors would like to thank Dr. R. Hanssen from Organon, (The Netherlands) for providing recombinant human FSH. We also thank Mr. H. Heuveling for the supply of ovaries.
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[11] Fraser LR. Albumin is required to support the acrosome reaction but not capacitation in mouse spermatozoa in vitro. J Reprod Fertil 1985;74:185±96. [12] Itagaki Y, Toyoda Y. Factors affecting fertilization in vitro of mouse eggs after removal of cumulus oophorus. J Mammal Ova Res 1991;8:126±34. [13] Siddiquey AKS, Cohen J. In vitro fertilization in the mouse and the relevance of different sperm/egg concentrations and volumes. J Reprod Fertil 1982;66:237±42. [14] Bavister BD. Evidence for a role of post-ovulatory cumulus components in supporting fertilizing ability of hamster spermatozoa. J Androl 1982;3:365±72. [15] Kikuchi K, Nagai T, Motlik J, Shioya Y, Izakie Y. Effect of follicle cells on in vitro fertilization of pig follicular oocytes. Theriogenology 1993;39:593±9. [16] Nandi S, Chauhan MS, Palta P. In¯uence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro. Theriogenology 1998;50:1251±62. [17] Hunter RHF. The fallopian tubes: their role in fertility and infertility. Springer-Verlag: Berlin, 1988. [18] Schroeder AC, Eppig JJ. The developmental capacity of mouse oocytes that matured spontaneously in vitro is normal. Dev Biol 1984;102:493±7. [19] Cox JF, Hormazabal J, Santa Maria A. Effect of cumulus on in vitro fertilization of bovine matured oocytes. Theriogenology 1993;40:1259±67. [20] Crozet N. Ultrastructural aspects of in vivo fertilization in the cow. Gamete Res 1984;10:241±51. [21] Chian RC, Okuda K, Niwa K. In¯uence of cumulus cells on in vitro fertilization of bovine oocytes derived from in vitro maturation. Anim Reprod Sci 1995;38:37±48. [22] Fukui Y. Effect of follicle cells on acrosome reaction, fertilization and developmental competence of bovine oocytes matured in vitro. Mol Reprod Dev 1990;26:40±6. [23] Ijaz A, Lambert RD, Sirard MA. In vitro-cultured bovine granulosa and oviductal cells secrete sperm motility-maintaining factor(s). Mol Reprod Dev 1994;37:54±60. [24] Downs SM, Schroeder AC, Eppig JJ. Serum maintains the fertilizability of mouse oocytes matured in vitro by preventing hardening of the zona pellucida. Gamete Res 1986;15:115±22. [25] Katska L, Kauffold P, Smorag Z, Duschinski V, Torner H, Kanitz W. In¯uence of hardening of the zona pellucida on in vitro fertilization of bovine oocytes. Theriogenology 1989;32:767±77. [26] Mattioli M, Galeati G, Seren E. Effect of follicle somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation. Gamete Res 1988;20:177±83. [27] Magier S, Van der Ven HH, Diedrich K, Krebs D. Signi®cance of cumulus oophorus in vitro fertilization and oocyte viability and fertility. Hum Reprod 1990;5:847±52. [28] Legendre LM, Stewart-Savage J. Effect of cumulus maturity on sperm penetration in the golden hamster. Biol Reprod 1993;49:82±8. [29] Behalova E, Greve T. Penetration rate of cumulus-enclosed versus denuded bovine eggs fertilized in vitro (abstract). Theriogenology 1993;39:186. [30] Cox JF. Effect of the cumulus cells on in vitro fertilization of in vitro matured cow and sheep oocytes (abstract). Theriogenology 1991;35:191. [31] Ball GD, Leibfried ML, Lenz RW, Ax RL, Bavister BD, First NL. Factors affecting successful in vitro fertilization of bovine follicular oocytes. Biol Reprod 1983;28:717±25. [32] Hawk HW, Nel ND, Waterman RA, Wall RJ. Investigation of means to improve rates of fertilization in vitro matured/in vitro fertilized bovine oocytes. Theriogenology 1992;38:989±98. [33] Parrish JJ, Susko-Parrish J, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988;38:1171±80. [34] Quirk SM, Fortune JE. Plasma concentration of gonadotrophins, pre-ovulatory follicular development and luteal function associated with bovine follicular ¯uid-induced delay of oestrus in heifers. J Reprod Fertil 1986;76:609±21. [35] Chian RC, Park CK, Sirard MA. Cumulus cells act as a sperm trap during in vitro fertilization of bovine oocytes (abstract). Theriogenology 1996;45:258. [36] Liu Y, Holyoak GR, Wang S, Bunch TD. The importance of cumulus cells on the in vitro production of bovine oocytes (abstract). Theriogenology 1995;43:267. [37] Li R, Norman RJ, Armstrong DT, Gilchrist RB. Oocyte-secreted factor(s) determine functional differences between bovine mural granulosa cells and cumulus cells. Biol Reprod 2000;63:839±45.
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[38] Uhler ML, Leung A, Chan SYW, Wang C. Direct effect of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil Steril 1992;58:1191±8. [39] Sirivaidyapong S, Bevers MM, Colenbrander B. Acrosome reaction in dog sperm is induced by a membrane-localized progesterone receptor. J Androl 1999;20:537±44. [40] Cheng FP, Gadella BM, Voorhout WF, Fazeli A, Bevers MM, Colenbrander B. Progesterone induced acrosome reaction in stallion spermatozoa is mediated by a plasma membrane progesterone receptor. Biol Reprod 1998;59:733±42.
Effect of cumulus cell removal of in vitro matured bovine oocytes prior to in vitro fertilization on subsequent cleavage rate A.N. Fatehia,*, E.C. Zeinstraa, R.V. Kooijb, B. Colenbrandera, M.M. Beversa a
Department of Farm Animal Health, Utrecht University, Utrecht, The Netherlands Centre for Reproductive Medicine, IVF-laboratory, Neusserstr 111, 40489 DuÈsseldorf, Germany
b
Received 7 June 2001; accepted 27 July 2001
Abstract The aim of this study is to identify the effect of cumulus cells removal prior to the in vitro fertilization of matured bovine oocytes on cleavage rate. Denuded, matured oocytes were fertilized in presence or absence of loose cumulus cells, cumulus cell conditioned IVF medium (CCCM), charcoal-treated CCCM and charcoal-treated CCCM supplemented with progesterone at a ®nal concentration of 150 ng/ml. After 18 h of incubation with sperm, the presumptive embryos were cultured on a BRL monolayer and the percentage of cleaved embryos was evaluated on Day 4. Removal of cumulus cells prior to IVF signi®cantly reduced the cleavage rate (25% for denuded oocytes versus 56% for cumulus±oocyte complexes (COCs)). The addition of loose cumulus cells partially restored the effect of denudation (cleavage rate: 37% for denuded oocytes supplemented with loose cumulus cells versus 27% for denuded oocytes and 58% for COCs). CCCM also had a positive effect on the cleavage rate of oocytes denuded prior to IVF (36% for denuded oocytes fertilized in CCCM versus 14% for denuded oocytes). Treating the CCCM with charcoal resulted in complete loss of its effect on cleavage rate (18% for denuded oocytes fertilized in charcoal-treated CCCM versus 34% for denuded oocytes fertilized in CCCM). The addition of progesterone to charcoal-treated CCCM partially restored the reduction of the cleavage rate caused by charcoal treatment (27% for denuded oocytes fertilized in charcoal-treated CCCM supplemented with progesterone versus 14% for denuded oocytes fertilized in charcoal-treated CCCM and 36% for denuded oocytes fertilized in CCCM). In conclusion, removal of cumulus cells prior to IVF adversely affects the cleavage rate through loss of a factor secreted by these cells. This factor probably is progesterone. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Cumulus cells; Progesterone; IVF; COC * Corresponding author. E-mail address: [email protected] (A.N. Fatehi).
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 1 7 - 8
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1. Introduction The importance of cumulus cells for the maturation and acquisition of developmental competence in oocytes has been well-established [1±5]. Removal of cumulus cells from the oocytes prior to in vitro maturation adversely affects the maturation, fertilization and embryo development in rat [6], sheep [7] and cattle [5,8]. In almost all species a certain proportion of cumulus cells are still present at the site of fertilization [9,10]. In vitro fertilization in the presence of cumulus cells increased the fertilization rate in mouse [11±13], hamster [14], pig [15] and buffalo [16]. The mechanism by which cumulus cells facilitate fertilization is not clear. It has been suggested that cumulus cells are involved in the interaction between male and female gametes, guiding spermatozoa towards the oocytes [17,18], inducing capacitation [19,20], acrosome reaction [21,22], maintaining sperm motility and viability [22,23], preventing zona pellucida from precocious hardening [24±26] and enhancing sperm penetration and in vitro fertilization [19,22,27,28]. In cattle, Behalova and Greve [29], Cox [30] and Ball et al. [31] showed that the presence of cumulus cells surrounding the oocytes is not necessary for successful IVF. They reported almost equal rates of sperm penetration during IVF of cumulus-free and cumulus-enclosed oocytes. Hawk et al. [32] observed a negative effect of cumulus cells on sperm penetration, while Younis and Brackett [4], Cox et al. [19], and Zhang et al. [5] reported a positive effect of cumulus cells on the in vitro fertilization of bovine oocytes. The aim of this study is to investigate if and how the cumulus cells affect the in vitro fertilization of bovine oocytes. 2. Materials and methods 2.1. Collection of oocytes Bovine ovaries were collected at a slaughterhouse in a thermo¯ask and transported to the laboratory within 1 h. Excised ovaries were rinsed in physiological saline (0.9% NaCl) containing antibiotics (100 IU penicillin and 100 mg streptomycin per ml) and dried with paper towel. Cumulus±oocyte complexes (COCs) were aspirated from small antral follicles (2±8 mm diameter) using an 18 gauage needle attached to a tube in line with a vacuum pump. The COCs were selected on the presence of a multilayered compact cumulus investment and homogeneous ooplasm and randomly assigned to the various treatments. 2.2. In vitro maturation, fertilization and embryo culture In vitro maturation and IVF as well as in vitro embryo culture (IVC) took place at 39 8C in a humidi®ed atmosphere of 5% CO2 in air. Selected COCs were rinsed in HEPES buffered M199, (Gibco BRL, Paisely, UK) supplemented with 10% fetal calf serum (FCS). Groups of 35 were randomly allocated to each well of a 4 well culture plate (Nunc A/S, Roskilde, Denmark) containing 500 ml of maturation medium per well. They were then cultured for 23 h. Unless otherwise indicated the maturation medium consisted of tissue
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culture medium M199 (Gibco, Life Technologies, Breda, The Netherlands) supplemented with 10% FCS and 0.05 units/ml recombinant human FSH (Organon, Oss, The Netherlands). Frozen/thawed spermatozoa used for IVF were centrifuged over a percoll gradient for 30 min at 700 g at 25 8C. The sperm sample was collected by removing the gradient for the last 150 ml containing the sperm pellet. Thirty-®ve COCs or denuded oocytes were transferred to 430 ml of fertilization medium (Fert-TALP) as described by Parrish et al. [33] without glucose and 10 mg/ml penicillin/streptomycin instead of gentamycin. We added to each well 20 ml of sperm suspension (®nal concentration 0.5106 spermatozoa/ml), 20 ml heparin (®nal concentration 10 mg/ml) and 20 ml PHE (consisting of 20 mM D-penicillamin, 10 mM hypotaurine, and 1 mM epinephrine). After 18±20 h of incubation, the presumptive zygotes were freed from cumulus cells by vortexing for 3 min and the groups were randomly placed in a co-culture system of 0.5 ml M199 supplemented with 10% FCS on a monolayer of buffalo rat liver (BRL) cells in each well of a 4 well culture plate. On the fourth day of culture, the proportion of cleaved embryos was assessed. 2.3. BRL cell culture Buffalo rat liver (BRL) cells separated from the BRL cell line from the American Type Culture Collection (ATCC) were routinely cultured in a 1:1 mixture of Ham's F12 medium and Dulbecco's modi®ed Eagle's medium (Gibco) supplemented with 7.5% FCS (Gibco) and antibiotics. 2.4. Preparation of IVF conditioned medium A total of 23 h after incubation of 140 COCs in maturation medium, the attached cumulus cells were removed by vortexing for 3 min. The detached cumulus cells were recovered, then divided in to each well of a 4 well culture plate containing 500 ml of IVF medium. The suspension was incubated at 39 8C in 5% CO2 for 18 h. After incubation, the supernatant was ®ltered through a 0.22 mm ®lter (Millipore, Bedford, MA, USA). The ®ltrate was designated as cumulus cell conditioned medium (CCCM) and was used the same day as preparation. 2.5. Charcoal treatment of conditioned medium To remove small molecules including steroids [34], part of the CCCM was stirred for 10 min with 50 mg charcoal/ml (Norit: activated and neutralized; Sigma St. Louis, MO, USA) and then centrifuged at 4500 g for 10 min at room temperature. The supernatant was centrifuged again at 11; 600 g for 3 min and subsequently ®ltered through a 0.22 mm ®lter (Millipore) to remove charcoal particles. The ®ltered supernatant was designated as charcoal-treated CCCM. 2.6. Preparation of progesterone solution A stock solution of progesterone was freshly prepared by dissolving 0.15 mg progesterone (Sigma) in 1 ml DMSO. Using the stock solution, a solution of charcoal-treated
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CCCM supplemented with progesterone at a ®nal concentration of 150 ng/ml was prepared. The ®nal concentration of DMSO in this medium was 0.1% (v/v). 2.7. Experiments A series of ®ve experiments were carried out. Unless otherwise indicated IVM, IVF, and IVC were performed according to the procedures above. 2.7.1. Experiment 1 COCs were in vitro matured and the cumulus cells were subsequently removed. Denuded oocytes were in vitro fertilized and after IVF, the presumptive zygotes were cultured as described. Cultures in which COCs were stripped after IVF served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of nine replicates. 2.7.2. Experiment 2 COCs were in vitro matured and the cumulus cells were subsequently stripped. Denuded oocytes were in vitro fertilized in the presence or absence of the detached cumulus cells. Cultures in which COCs were stripped after IVF served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of three replicates. 2.7.3. Experiment 3 COCs were in vitro matured and the cumulus cells were subsequently stripped. Denuded oocytes were in vitro fertilized in CCCM. Denuded oocytes fertilized in plain IVF medium served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of four replicates. 2.7.4. Experiment 4 COCs were in vitro matured and the cumulus cells were subsequently stripped. Denuded oocytes were in vitro fertilized in charcoal-treated CCCM. Denuded oocytes fertilized in plain IVF medium served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of ®ve replicates. 2.7.5. Experiment 5 COCs were in vitro matured and the cumulus cells were subsequently stripped. Denuded oocytes were in vitro fertilized in charcoal-treated CCCM supplemented with progesterone. Denuded oocytes, fertilized in CCCM or charcoal-treated CCCM served as control. The percentage of cleaved embryos 4 days after fertilization was assessed. The experiment consisted of ®ve replicates. 2.8. Statistical analysis The results were analyzed by Chi-square test and P < 0:05 was considered signi®cant.
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3. Results The removal of cumulus cells before in vitro fertilization of in vitro matured oocytes signi®cantly decreased the cleavage rate. (Table 1). When cumulus cells were removed by a narrow-bore pasteur pipette, instead of vortexing, a similar decrease of the cleavage rate was observed (data not shown). Addition of removed cumulus cell to the IVF medium of denuded oocytes partially restored the denudation effect and increased the cleavage rate (Table 2). In vitro fertilization of denuded oocytes in CCCM increased the cleavage rate (Table 3) as compared to the cleavage rate of denuded oocytes routinely in vitro fertilized in IVF medium. Treatment of CCCM with charcoal, resulted in a complete disappearance of its promoting effect on the cleavage rate of denuded oocytes following fertilization (Table 4). Table 1 The effect of cumulus cell removal before in vitro fertilization of in vitro matured bovine oocytes on cleavage rate
Cumulus-enclosed oocytes Cumulus-free oocytes
Oocytes (n)
Cleavage rate Day 4 (%)
1222 1092
684 (56) a 270 (25) b
a and b: P < 0:0001. Table 2 The effect of loose cumulus cells on the in vitro fertilization of denuded bovine oocytes
Cumulus-enclosed oocytes Denuded oocytes Denuded oocytes loose cumulus cells
Oocytes (n)
Cleavage rate Day 4 (%)
263 201 262
155 (58) a 54 (27) b 98 (37) c
a,b and a,c: P < 0:0001; b,c: P < 0:05. Table 3 The effect of cumulus cell conditioned medium (CCCM) on the cleavage rate of bovine oocytes denuded prior to IVF
Denuded oocytes Denuded oocytes CCCM
Oocytes (n)
Cleavage rate Day 4 (%)
640 479
95 (14) a 171 (36) b
a and b: P < 0:0001. Table 4 The effect of charcoal treatment of cumulus cell conditioned medium (CCCM) on the cleavage rate of bovine oocytes, denuded prior to IVF
Denuded oocytes Denuded oocytes CCCM Denuded oocytes charcoal-treated CCCM a and b: P < 0:0001.
Oocytes (n)
Cleavage rate Day 4 (%)
533 565 580
102 (19) a 193 (34) b 105 (18) a
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Table 5 The effect of charcoal-treated cumulus cell conditioned medium (CCCM) supplemented with progesterone on the cleavage rate of bovine oocytes denuded prior to IVF Denuded oocytes fertilized in
Oocytes (n)
Cleavage rate Day 4 (%)
CCCM Charcoal-treated CCCM Charcoal-treated CCCM progesterone
476 459 509
172 (36) a 66 (14) b 139 (27) c
a,b and b,c: P < 0:0001, a,c: P < 0:05.
Adding progesterone (150 ng/ml) to charcoal-treated CCCM partially restored the lost effect and resulted in a higher cleavage rate of bovine oocytes denuded prior to IVF (Table 5). 4. Discussion The results demonstrate that the presence of cumulus cells has a positive in¯uence on the in vitro fertilization of bovine oocytes. The cleavage rate signi®cantly decreases when cumulus cells are removed prior to in vitro fertilization. These results are in agreement with previous reports showing that the presence of cumulus cells facilitates fertilization of cattle oocytes leading to a higher penetration rate [19,21,35], higher cleavage rate [4,5,22,36] and a higher yield of blastocysts [5,36]. In other studies [29,30,31], it was found that removal of cumulus cells did not affect the penetration rate. However, male and female pronucleus formation was impaired [31] and a higher incidence of polyspermia was observed [29]. Cox et al. [19] reported that the positive effect of cumulus cells on fertilization is expressed only when the cells are in contact with the zona pellucida. In contrast, we found that the presence of loose cumulus cells partially restored the effect of denudation prior to in vitro fertilization. This suggests that both the presence of cumulus cells and cell±oocyte contact contribute to a proper fertilization process. The partial restoration of the fertilization rate might be due to the fact that the mechanical procedure of cumulus cell removal has harmed part of the oocytes, resulting in lower male pronuclear formation as suggested by Ball et al. [31]. The increase of cleavage rate of denuded oocytes by CCCM suggests that the positive effect of cumulus cells is the result of compound(s) secreted by cumulus cells. The fact that charcoal treatment inactivates the bene®cial effect of CCCM indicates that the secreted factor is a small molecule [33]. Progesterone probably is the active molecule since the addition of progesterone to charcoal-treated CCCM increased the fertilization rate of denuded oocytes. The lower fertilization rate of charcoal-treated CCCM supplemented with progesterone as compared to nontreated CCCM might be a concentration effect or may suggest that apart from progesterone, another small molecule is involved. Evidence that progesterone is the active molecule secreted by cumulus cells is supported by the study of Li et al. [37] which shows that COCs from 2 to 6 mm follicles produce progesterone. In our study, loose cumulus cells and CCCM almost equally increased the cleavage rate of denuded oocytes, suggesting that the amount of secreted progesterone was not signi®cantly
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different. Li et al. [37] observed a higher progesterone production by oocytectomized cumulus±oocyte complexes in the presence of IGF and FSH. As CCCM is prepared by the incubation of cumulus cells in routine fertilization medium, thus without IGF and FSH, the production of progesterone probably was not enhanced due to the absence of oocytes. A number of reports indicate that progesterone affects various sperm functions. Progesterone stimulates hyperactivity in human sperm [38] and increases the acrosome reaction in human [38], dog [39] and stallion sperm [40]. Moreover, progesterone in vitro enhances the number of stallion spermatozoa that binds to the zona pellucida [40]. In the case of dog [13] and stallion sperm [40], it has been shown that the effect of progesterone is exerted through a progesterone receptor localized in the plasma membrane of spermatozoa. Hence, it can be assumed that the positive effect of cumulus cells on the in vitro fertilization of bovine oocytes is at least partially caused by progesterone secreted by the cumulus cells. In conclusion, this study suggests that removal of cumulus cells from in vitro matured oocytes, prior to in vitro fertilization adversely affect the cleavage rate of cattle oocytes by the elimination of progesterone secreted by the cumulus cells.
Acknowledgements Grant sponsor: EU Commission; Grant number QLK3-CT1999-CT00104. The authors would like to thank Dr. R. Hanssen from Organon, (The Netherlands) for providing recombinant human FSH. We also thank Mr. H. Heuveling for the supply of ovaries.
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