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Role of EGF, IGF-I, sera and cumulus cells on maturation in vitro of bovine oocytes
ROLE OF EGF, IGF-I, SERA AND CUMULUS CELLS ON MATURATION IN VITRO OF BOVINE OOCYTES P.L. Lorenzo, Departamento
M.J. Illera, J.C. Illera and M. Illera’
de Fisiologia Animal. Facultad de Veterinaria. Ciudad Universitaria. 28040. Madrid, Spain
Received
for publication: Accepted:
UCM
May 16, 1994 December 2, 1994
ABSTRACT We examined the effects of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), the presence/absence of cumulus cells, and sera (fetal calf serum, FCS or estrous cow serum, ECS) on cumulus expansion and meiotic maturation during in vitro bovine oocyte maturation. The oocytes obtained (n= 4491) were divided into cumulusoocyte complexes (COC) or denuded oocytes and were then cultured in 3 groups comprising TCM-199 and sera: Group I (without serum), Group II (10% FCS), and Group III (10% ECS). Each group was subjected to 4 predefined treatments with growth-factors: control (no growth factor), EGF, IGF-I, and EGF+IGF-I. At the end of the 24-h culture period, the oocytes were assessed for degree of cumulus expansion and metaphase II stage. Treatments with EGF enhanced the incidence of full cumulus expansion in all 3 groups. Maximal stimulation for both cumulus expansion and nuclear maturation occurred with EGF + IGF-I in all groups, m’ain ly in the groups with serum. Regarding the denuded oocytes, no positive effects on nuclear maturation rates were observed for any treatment. These results suggest that: 1) EGF, with or without IGF-I, stimulates cumulus expansion and meiotic maturation significantly; and 2) the presence of FCS or ECS enhances the effect of these growth factors in bovine cumulus-oocyte complexes. Key words:
EGF, IGF-I, bovine oocyte, cumulus expansion,
nuclear
maturation
INTRODUCTION The mammalian oocyte is arrested at the dyctiate stage of the meiotic prophase, until shortly before ovulation, when the preovulatory gonadotrophin surge triggers the resumption of the meiotic process. The preovulatory luteinizing hormone (LH) surge is generally accepted as the endocrine process regulating induction of in vivo oocyte maturation, since exposure of the follicle to LH or human chorionic gonadotropin (hCG) induces maturation (18). Completion of maturation ismorphologically identified by metaphase II stage (M II), and is accompanied by expansion of the layers of the cumulus cellssurrounding the oocyte. imply that LH is only one of a complex sequence of factors More recent observations Acknowledgments: Ovaries were generously supplied by Dr. P. Jimenez (GYPISA). We thank Dra. G. Silvan, Dr. J. Sanchez and T. Calduch for help in preparing this manuscript and assistance, and F. Pescador for advice with statistical analysis. ’ Correspondence and reprint requests. Theriogenology 447 09-l 16, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691 x/95/$1 0.00 SSDI 0093-691 X(95)001 52-X
110
Theriogenology
involved in oocyte maturation. Studies with rodents have implicated some growth factors in meiotic maturation. In vitro studies with EGF showed induction of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes maintained in meiotic arrest with purines, dbc-AMP, or the phosphodiesterase inhibitor IBMX (7,8). Epidermal growth factor has also been shown to stimulate murine cumulus expansion in vitro (8). It has also been demonstrated that IGF-I participates in the regulation of many ovarian functions; for example, it is a potent mitogen for granulosa cells (17) and acts as a biological amplifier of FSH action in the ovary (20). However, there is very little information about growth-factor induced regulation of oocyte maturation and cumulus expansion in domestic species. Furthermore, serum supplementations of maturation media have been regularly used in in vitro maturation systems to give a protein and energy source to the oocyte during maturation. Although it is fetal calf serum (FCS) that has been most frequently used (22), Sanbuissho and Threlfall (26) point out that utilization of estrous cow serum (ECS) increases oocyte maturation and fertilization capacity. We set out to examine the effects of EGF, IGF-I and serum supplementation on the completion of meiotic maturation and cumulus expansion in bovine oocytes in vitro. The results obtained may serve to shed light on some physiological functions of these growth factors with respect to bovine oocyte maturation. MATERIALS
AND METHODS
In Vitro Oocyte Maturation Bovine ovaries were obtained from the slaughterhouse (GYPISA, Pozuelo de Alar&n, Madrid) and were transported in PBS (Dulbecco’s phosphate buffer saline) to the laboratory within 1 h. Follicular contents from small antral follicles (2 to 8 mm in diameter) were aspirated with a 18-gauge needle attached to a lo-ml disposable syringe. The oocytes were transferred onto a 35-mm plastic Petri dish (Bibby, Stone, Staffordshire, England, cat. no. 2333) containing 2 ml of Hepes-buffered washing medium (TCM 199 Hepes-buffered, Sigma Chemical Co. St. Louis, MO, cat. no. 2520) supplemented with 2% FCS (Gibco Ltd, Paisley, Scotland, cat. no. 6290-H), followed by 5 washes. According to predefined criteria (23), selected cumulus-oocytes were divided for culturing either as cumulus-oocyte complexes (with intact and unexpanded cumulus: COC) or denuded oocytes (without layers of cumulus cells), placed into drops (50 ~1) of maturation medium under oil and cultured on 35-mm Petri dishes at 39°C in 5% CO*, 95% air and 100% relative humidity for 24 h. The oocytes (5 per drop) were matured in TCM-199 (Earle’s salt with sodium bicarbonate and L-glutamine, Sigma, cat. no. 4530), this basic medium being supplemented with sera to form 3 treatment groups: Group I (without serum), Group II (with 10% v/v FCS), and Group III (with 10% v/v ECS). Each group then underwent 4 different, predefined treatments with growth factors (Boehringer Mannheim, Germany): control (no growth-factor), EGF (50 ng/ml), IGF-I (100 ng/ml), and EGF+IGF-I (50 and 100 ng/ml, respectively), for a total of 12 maturation treatments. Sera were heat-inactivated (56°C for 30 min) before use. The ECS was obtained from cows during standing estrus and was pooled as a stock reserved for use in this study.
111
Theriogenology Cumulus
Expansion
Evaluation
Cumulus oocyte complexes were evaluated at the end of the culture period in order to assess the effect of growth factors on cumulus expansion (Experiment 1). The cumulus expansion was scored based on the subjective scale of 0 to 3, in which 0 indicates no detectable expansion; + 1 indicates the minimum observable response (outer 1 or 2 layers expanded); t 2 indicates outer one-half of the cumulus expanded; and +3 indicates all layers of cumulus cells expanded, even those closest to the oocyte. Nuclear
Maturation
Evaluation
At the end of the culture period, all the oocytes were fixed to ascertain the influence of growth factors on nuclear maturation in vitro, and the effect of the presence or absence of cumulus cells surrounding the oocyte (Experiment 2). Cumulus cells of COCs were removed with hyaluronidase (200 UI/ml) and were mechanically stripped using a fine-bore pipette. The oocytes were then pipetted onto a slide. A coverslip spotted with a paraffinVaseline (1O:l) mixture at each corner was placed directly over the center of the drop containing the oocytes. Fixation of oocytes was carried out by placing the slides in acetic acid-ethanol (1:3) for 24 h and staining with aceto-orcein (2% orcein in 60% acetic acid) for 2 min. Nuclear maturation was evaluated and classified as germinal vesicle (GV), metaphase I (M I), metaphase II (M II) and degeneration. Statistical
Analysis
Other than the cumulus expansion experiment, which was performed 5 times, the nuclear maturation experiment was replicated 7 times. Statistical analysis using the Chisquare test was carried out in pooled data using the Biomedical Data Program (6); P values less than 0.05 were considered to be significant. Maximum cumulus expansion degree in Experiment 1 and Metaphase II stage in Experiment 2 were used as end-point parameters for assessing growth-factor and sera effect on bovine oocyte maturation. Data are present in results as percent mean r SEM of oocytes having undergone full expansion degree in Experiment 1 or M II stage in the Experiment 2. RESULTS Experiment
1
A total of 1105 bovine cumulus-oocyte complexes were used and evaluated in this experiment. Data for those COCs which attained full cumulus expansion are shown in Table 1. Compared with control and IGF-I treatments, EGF treatments resulted in a significantly higher incidence of cumulus expansion in Groups I and II (PcO.05) though not in Group III. When EGF was used in conjunction with IGF-I, cumulus expansion registered higher readings in all groups, these data being significantly higher than for treatments with IGF-I alone. No significant differences were observed between treatments with IGF-I and those without growth factors. In no case did COCs exhibit full expansion when cultured without growth factors and serum.
112
Theriogenology
Table 1. Cumulus expansion observed in bovine cumulus-oocyte complexes in response to various combinations of growth factors (EGF, 50ng/ml; IGF-I, 100 ng/ml; EGF+IGF-I, 50 and 100 ng/ml) and sera (none, FCS and ECS) after 24 hours of in vitro maturation Cumulus expansion % (observed/total oocytes) Treatment
Group I (Without serum)
Group II (10% FCS)
Group III (10% ECS)
TCM-199
0% (0/79)a
38.5% (32/83)a
44.8% (39/87)a
TCM-199+EGF
49.4% (45/91)h
62.6% (57/91)b
57.3% (51/89)a
TCM-199 + IGF-I
10.5% (10/95)a
40.6% (41/101)a
54.0% (53/98)a
TCM199 + EGF + IGF-I
55.3% (52/94)b
74.2% (69/93)bYc
75.9% (79/H@
Data were pooled from 5 independent experiments. Means are expressed (full expansion/total number of oocytes). Values with different superscripts significantly different: a vs b, PcO.05; a vs c, P< 0.01 by Chi-square test.
Experiment
as percentages in columns are
2
Oocyte totals, fixed and stained for maturity (n= 3386), are depicted in Figure 1 (COC) and Figure 2 (denuded oocytes). With regard to COCs, the highest metaphase II values in Group I (without serum) were recorded in response to EGF+IGF-I treatment (60.0 f 3.1%) and proved to be significantly different (PcO.05) from those recorded for IGF-I and control treatments. In Group II (with 10% FCS) the best maturation result was obtained with EGF+IGF-I (77.5 f 2.2%), there being significant differences compared with TCM199+FCS (41.6 f 4-l%, PcO.01) and IGF-I treatments (54.1 + 2.1%, PcO.05) but not with EGF treatment. Similarly, Group III (with 10% ECS) showed higher maturation means in response to EGF+ IGF-I treatment (75.8 k 2.3%), revealing statistical differences (P < 0.01) with TCM-199+ECS treatment. Nuclear maturation rates for treatments of all groups with serum were greater than for the group without. As between Groups II and III (both with serum), no differences were observed in treatments except when using IGF-I, which increased the maturation rate in the ECS-group compared with that of the FCS-group (64.5 f 2.3% vs 54.1 k 2.1%, PcO.05).
113
Theriogenology % of M II
**
**
80 70 60 50
n
NONE
EGF
IGF-I
(ill)
(148)
(113)
GROUP Figure 1.
No rate of the maturation treatments
I
EGF+IGF NONE (180)
(144)
EGF
IGF-I EGF+IGF NONE
EGF
IGF-1 EGF+IGF
(170)
(168)
(116)
(ZOO)
GROUP
III
GROUP
II
(156)
(141)
(207)
The effect of growth factors and sera on meiotic maturation of bovine cumulus-oocyte complexes. Oocytes were cultured in medium containing no added serum (Group I), 10% FCS (Group II), and 10% ECS (Group III) as well as with different growth factors (no growth factor; EGF, 50 ng/ml; IGF-I, 100 ng/ml, and EGF+IGF-I, 50 and 100 ng/ml). Metaphase II stage was assessed after 24 hours of culture. Data represent percentage means 2 SEM of 9 independent experiments. Bars with an asterisk indicate significant difference with the control (* P < 0.05; * * P < 0.01). significant differences among treatments were observed regarding the maturation denuded oocytes (P > 0.05, Figure 2). Cumulus-oocyte complexes exhibited higher values than denuded oocytes in all treatments, except for the control and IGF-I in Groups I and II.
DISCUSSION Immature oocyte development in the ovarian follicle to mature oocyte (metaphase II stage) depends on many regulatory factors (31,32). Cumulus oophorus expansion in bovine oocytes occurs in response to an ever-changing milieu of gonadotrophins, growth factors, steroids, factors secreted by the oocyte and other unknown molecules (3). These compounds could be contributing to maturational changes that occur in the oocyte, mediated by intracellular messengers such as CAMP, calmodulin or diacylglycerol (12). There are some criteria evaluating expansion of cumulus cells, and in the present study, we adopted similar expansion criteria to those used by other authors (3,8,29).
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Theriogenology
% of M II 80 70 60 50
f
+ 40 30 20 10
-
-
0
n
NONE
EGF
IGF-I
EGF+IGF
NONE
EGF
IGF-I
(88)
wx
(171)
(144)
(103)
(148)
(114)
GROUP Figure 2.
I
GROUP
II
EGF+IGF (140)
NONE
EGF
IGF-I
(109)
(99)
(11’5)
GROUP
1 EGF+IGF (140)
III
The effect of growth factors and sera on meiotic maturation of bovine denuded oocytes. Oocytes were cultured in medium containing no added serum (Group I), 10% FCS (Group II), and 10% ECS (Group III), as well as with different growth factors (no growth factor; EGF, 50 ng/ml; IGF-I, 100 ng/ml, and EGF + IGF-I, 50 and 100 ng/ml). Metaphase II stage was assessed after 24 hours of culture. Data represent percentage means + SEM of 9 independent experiments. Bars with an asterisk indicate significant difference with the control. No significant differences with the control were observed.
Use was made of a serum-free group media (Group I) for in vitro maturation in order to explain the relationship between growth factors and the regulation of nuclear maturation and cumulus expansion, while effectively ruling out the influence of unknown serum factor (s). The results obtained indicate that EGF enhances cumulus expansion in bovine COCs in the same way as demonstrated by Downs (8) for murine oocytes. However, the lesser effect of EGF on bovine cumulus expansion compared to that seen in mouse oocytes may be due to the fact that, whereas recombinant human EGF was used in this study, homologous murine EGF was used in Downs’ mouse study (8). Although IGF-I per se is unable to promote effective cumulus expansion, this did not impair the maturation rate. Since IGF-I receptors have been detected in granulosa cells and oocytes (2), it is possible that suppression of expansion may be mediated by IGF-I acting on the granulosa cells directly or inhibiting the production of an expansion factor produced by the oocyte itself (3).
Theriogenology
115
Other authors using IGF-I (16) or IGF-I analogues, such as insulin (36), likewise recorded poor results for bovine cumulus expansion. The greater effect exerted by growth factors plus serum (FCS or ECS) on bovine cumulus expansion can be explained on the basis of the hormonal content of such sera. Both FCS and ECS are routinely used in culture media for oocyte maturation (10,11,22,27), yet they differ in the comparatively greater concentrations of LH, TSH and estradiol-170 present in ECS (33,34). Addition of sera to growth-factor media has an effect on EGF and EGF+IGF-I induced cumulus expansion, suggesting that these compounds may be acting in the cumulus cells through a synergic mechanism. In the present study, both EGF and EGF+IGF-I combined, with or without serum, effectively stimulated cumulus expansion. To date, however, there has been no study describing the interactions between these 2 growth factors with the aim of evaluating cumulus expansion. The highest percentages of EGF-induced nuclear maturation correspond to those obtained in rodent oocytes (58). Such high rates can be attributed to the fact that the main agent involved in the inhibition of meiotic resumption in these species’ oocytes is the stimulation of CAMP-dependent protein kinase. However, in their response to protein kinase stimulation via CAMP accumulation, bovine oocytes seem to differ from those of the above species (30), possessing other substances inhibiting meiotic activation (e.g., purines) upon which EGF, in all likelihood, fails to act in the same way. In our maturation experiment, the highest results were achieved with the addition of EGF+IGF-I, such data being in line with the recent results obtained in porcine oocytes (25). This could indicate that, under in vivo conditions, there may be an additive element at work in the way in which these two growth factors combine their respective actions. They bring about specific actions on oocyte maturation, including cumulus and cytoplasmic effects. Hainaut et al. (13) postulated that maturation with IGF-I is initiated upon activation of the membrane receptor for this growth factor and requires tyrosine dephosphorylation of ~34, the kinase component of maturation promoting factor (MPF). Otherwise, the IGF-I in the above cited reports, did not stimulate GVBD of mouse oocytes but did stimulate the resumption of meiosis for rat oocytes. In the present study, IGF-I, when acting singly, had a slight stimulatory effect on bovine-oocyte nuclear maturation. As between groups with sera, no significant differences in the case of maturation treatments were observed, except in the case of treatments using IGF-I alone; here, the maturation percentage was higher in the ECS- than in the FCS-group. IGF-I increases the number of receptors for LH in granulosa cells (1); hence, by using ECS, which has a higher LH concentration than FCS, nuclear maturation is boosted. These results were only observed in cumulus-oocyte complexes since LH needs cumulus cells if it is to act effectively on the oocyte (35). The obtained M II rates in COCs were low compared with those of other reports; these may have been due to variations in the FCS-commercial lots (10,28) or to the hormonal concentration of ECS (33). Ovarian and intrafollicular mechanisms for the regulation of oocyte growth and maturation are complex. The mechanisms whereby growth factors regulate or modulate resumption of meiosis in oocytes may be mediated via the granulosa and/or cumulus cells (4,5). It has been previously demonstrated that the growth growth factors can be synthesized by the ovary (14,24). The presence of EGF or EGF-like substances has been demonstrated for porcine and human follicles at concentrations similar to those used by us (19,21).
116
Theriogenology
Moreover, concentrations of IGF-I, similar to those used in the present study, have been detected in bovine and porcine follicular fluid (15). The growth factors and/or serum used in this study enhanced maturation in cumulus-oocyte complexes but not in denuded oocytes. Not only is this in accordance with the results observed in rodents (5,8) and those obtained using other growth factors such TGF-a (4), but it also demonstrates that growth-factor action requires the presence of cumulus cells, by which a positive stimulus for nuclear maturation is transferred to the oocyte. This paper provides evidence: 1) that inducer of meiotic maturation and cumulus presence of FCS or ECS enhances the effect and meiotic maturation in immature bovine
EGF, singly or combined with IGF, is a potent expansion in bovine oocytes; and 2) that the of these growth factors on cumulus expansion cumulus-oocyte complexes.
REFERENCES 1. Adashi EY, Resnick CE, Hernandez ER, Svoboda ME, Van Wyk JS. Characterization and regulation of a specific membrane receptor for somatomedinC/insulin-like growth factor-I in cultured rat granulosa cells. Endocrinology 1988; 112:194-201. 2. Balboni GC, Vanelli GB, Barni T, Orlando C, Serio M. Transferrin and somatomedin C receptors in the human ovary follicles. Fertil Steril 1987; 48:796 abstr. 3. Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ. FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte. Dev Biol 1990; 138:16-25. 4. Brucker C, Alexander NJ, Hodgen GD, Sandow BA. Transforming growth factor-a augments meiotic maturation of cumulus cell-enclosed mouse oocytes. Mol Reprod Dev 1991; 28:94-98. 5. Dekel N, Scherizly I. Epidermal growth factor induces maturation of rat follicle enclosed oocytes. Endocrinology 1985; 116:512-516. 6. Dixon WJ, Brown MB, Engelman L, Hill MA, Jennrich RI. BMDP Statistical Software Manual. Los Angeles: University of California Press, 1990. 7. Downs SM, Daniel SAJ, Eppig JJ. Induction of maturation in cumulus cell-enclosed mouse oocytes by follicle-stiomulating hormone and epidermal growth factor: evidence for a positive stimulus of somatic cell origin. J Exp Zoo1 1988; 245:86-96. 8. Downs SM. Specificity of epidermal growth factor action on maturation of the murine oocyte and the cumulus oophorus in vitro. Biol Reprod 1989; 41:371-379. 9. Echternkamp SE, Spicer LJ, Gregory KE, Channing SF, Hammond JM. Concentrations of insulin-like growth factor-I in blood and ovarian follicular fluid of cattle selected for twins. Biol Reprod 1990; 43:8-14. 10. Fukui Y. Effects of sera and steroid hormones on development of bovine oocytes matured and fertilized in vitro and co-cultured with bovine oviduct epithelial cells. J Anim Sci 1989; 67:1318-1323. 11. Fukui Y. Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro. Mol Reprod Dev 1990; 26:40-46.
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12. Goncalves PB, Graves CN. Interaction between CAMP, diacylglycerol and Ca”calmodulin pathways in the regulation of cumulus expansion. Biol Reprod 1992; 46 (Suppl. 1):69 abstr. 13. Hainaut P, Giorgetti S, Kowlaski A, Ballotti R, Van Obberghen E. Antibodies to phosphotyrosine injected to xenopus laevis oocytes modulate maturation induced by insulin/IGF-I. Exp Cell Res 1991; 195129-136. 14. Hammond JM, Baranao JLS, Skaleris D, Knight AB, Romanus JA, Rechler MM. Production of insulin-like growth factors by ovarian granulosa cell. Endocrinology 1985; 117:2553-2555. 15. Hammond JM, Hsu CJ, Klindt J, Tsang BK, Downey BR. Gonadotropins increase concentrations of immunoreactive insulin-like growth factor-I in porcine follicular fluid in vivo. Biol Reprod 1988; 38:304-308. 16. Herrler A, Lucas-Hann A, Niemann A. Effects of insulin-like growth factor-I on in vitro production of bovine embryos. Theriogenology 1992; 37:1213-1224. 17. Hernandez ER, Resnick CE, Svoboda ME, Van Wyk JJ, Payne DW, Adashi EY. Somatomedin-C/insulin-like growth factor I as an enhancer of androgen biosynthesis by cultured rat ovarian cells. Endocrinology 1988; 122:1603-1612. 18. Hillensjo T, Ekholm C, Ahren K. Role of cylic AMP in oocyte maturation and glycolysis in the pre-ovulatory rat follicle. Endocrinology 1978; 122:377-388. 19. Hoffmann GE, Scott RT Jr, Brzyski RG, Jones HW Jr. Immunoreactive epidermal growth factor concentration in follicular fluid obtained from in vitro fertilization. Fertil Steril 1990; 54:303-307. 20. Hsu C, Hammond JM. Gonadotropins and estradiol stimulate immunoreactive insulinlike growth factor-I production by porcine cells in vitro. Endocrinology 1987; 120:198207. 21. Hsu CJ, Holmes SD, Hammond JM. Ovarian epidermal growth factor-like activity: concentrations in porcine follicular fluid during follicular enlargement. Biochem Biophys Res Commun 1987; 147( 1):242-247 22. Leibfried-Rutledge ML, Critser ES, First NL. Effect of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulusoocyte complexes. Biol Reprod 1986; 35:850-857. 23. Lorenzo P, Illera MJ, Illera JC, Illera, M. Acciones especificas de 10s factores de crecimiento (EGF e IGF-I) en la maduracion in vitro de oocitos bovinos. Rev Esp Fisiol 1993; 49:265-270. 24. Makris A, Klagsbrum MA, Yasaumizu T, Ryan KJ. An endogenous ovarian growth factor with stimulates BALB/3T3 and granulosa cell proliferation. Biol Reprod 1983; 29:1135-1141. 25. Reed ML, Estrada JL, Illera MJ, Petters RM. Effects of epidermal growth factor, insulin-like growth factor-I, and dialyzed porcine follicular fluid on porcine oocyte nuclear maturation in vitro. J Exp Zoo1 1993; 266:74-78. 26. Sanbuissho A, Threlfall WR. The effect of estrous cow serum on the maturation and fertilization of the bovine follicular oocyte in vitro. Theriogenology 1989; 31:693-699. 27. Schellander K, Fuhrer G, Brackett BG, Korb H, Schleger W. In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum. Theriogenology 1990; 33:477-485. 28. Shiigi SM, Mishell RI. Sera and the in vitro induction of inmune responses. I. Bacterial contamination and generation of good fetal bovine sera. J Inmunol 1975; 115:741 abstr.
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29. Singh B, Barbe CJ, Armstrong DT. Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro. Mol Reprod Dev 1993; 36:113-119. 30. Sirard MA, Coenen K, Bilodeau S. Effects of fresh or cultured follicular fractions on meiotic resumption in bovine oocytes. Theriogenology 1992; 37:39-58. 31. Tonetta SA, DiZerega GS. Intragonadal regulation of follicular maturation, Endocrine Rev 1989; l&205-229. 32. Wassarman PM. The mammalian ovum. In: Knobil E, Neil1 JD (eds), The Physiology of Reproduction. New York: Raven Press, 1988;69-102. 33. Younis AI, Brackett BG, Fayrer-Hosken RA. Influence of serum and hormone on bovine oocyte maturation and fertilization in vitro. Gamete Res 1989; 23:189-201. 34. Younis AI, Brackett BG. Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro. Mol Reprod Dev 1992; 31:144-151. 35. Zuelke KA, Brackett BG. Effect of luteinizing hormone on glucose metabolism in cumulus enclosed bovine oocytes matured in vitro. Biol Reprod 1992; 46(Suppl. 1):117 abstr. 36. Zhang L, Blakewood EG, Denninston RS, Godke RA. The effect of insulin on maturation and development of in vitro-fertilized bovine oocytes. Theriogenology 1991; 35:301 abstr.
M.J. Illera, J.C. Illera and M. Illera’
de Fisiologia Animal. Facultad de Veterinaria. Ciudad Universitaria. 28040. Madrid, Spain
Received
for publication: Accepted:
UCM
May 16, 1994 December 2, 1994
ABSTRACT We examined the effects of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), the presence/absence of cumulus cells, and sera (fetal calf serum, FCS or estrous cow serum, ECS) on cumulus expansion and meiotic maturation during in vitro bovine oocyte maturation. The oocytes obtained (n= 4491) were divided into cumulusoocyte complexes (COC) or denuded oocytes and were then cultured in 3 groups comprising TCM-199 and sera: Group I (without serum), Group II (10% FCS), and Group III (10% ECS). Each group was subjected to 4 predefined treatments with growth-factors: control (no growth factor), EGF, IGF-I, and EGF+IGF-I. At the end of the 24-h culture period, the oocytes were assessed for degree of cumulus expansion and metaphase II stage. Treatments with EGF enhanced the incidence of full cumulus expansion in all 3 groups. Maximal stimulation for both cumulus expansion and nuclear maturation occurred with EGF + IGF-I in all groups, m’ain ly in the groups with serum. Regarding the denuded oocytes, no positive effects on nuclear maturation rates were observed for any treatment. These results suggest that: 1) EGF, with or without IGF-I, stimulates cumulus expansion and meiotic maturation significantly; and 2) the presence of FCS or ECS enhances the effect of these growth factors in bovine cumulus-oocyte complexes. Key words:
EGF, IGF-I, bovine oocyte, cumulus expansion,
nuclear
maturation
INTRODUCTION The mammalian oocyte is arrested at the dyctiate stage of the meiotic prophase, until shortly before ovulation, when the preovulatory gonadotrophin surge triggers the resumption of the meiotic process. The preovulatory luteinizing hormone (LH) surge is generally accepted as the endocrine process regulating induction of in vivo oocyte maturation, since exposure of the follicle to LH or human chorionic gonadotropin (hCG) induces maturation (18). Completion of maturation ismorphologically identified by metaphase II stage (M II), and is accompanied by expansion of the layers of the cumulus cellssurrounding the oocyte. imply that LH is only one of a complex sequence of factors More recent observations Acknowledgments: Ovaries were generously supplied by Dr. P. Jimenez (GYPISA). We thank Dra. G. Silvan, Dr. J. Sanchez and T. Calduch for help in preparing this manuscript and assistance, and F. Pescador for advice with statistical analysis. ’ Correspondence and reprint requests. Theriogenology 447 09-l 16, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691 x/95/$1 0.00 SSDI 0093-691 X(95)001 52-X
110
Theriogenology
involved in oocyte maturation. Studies with rodents have implicated some growth factors in meiotic maturation. In vitro studies with EGF showed induction of germinal vesicle breakdown (GVBD) in cumulus cell-enclosed mouse oocytes maintained in meiotic arrest with purines, dbc-AMP, or the phosphodiesterase inhibitor IBMX (7,8). Epidermal growth factor has also been shown to stimulate murine cumulus expansion in vitro (8). It has also been demonstrated that IGF-I participates in the regulation of many ovarian functions; for example, it is a potent mitogen for granulosa cells (17) and acts as a biological amplifier of FSH action in the ovary (20). However, there is very little information about growth-factor induced regulation of oocyte maturation and cumulus expansion in domestic species. Furthermore, serum supplementations of maturation media have been regularly used in in vitro maturation systems to give a protein and energy source to the oocyte during maturation. Although it is fetal calf serum (FCS) that has been most frequently used (22), Sanbuissho and Threlfall (26) point out that utilization of estrous cow serum (ECS) increases oocyte maturation and fertilization capacity. We set out to examine the effects of EGF, IGF-I and serum supplementation on the completion of meiotic maturation and cumulus expansion in bovine oocytes in vitro. The results obtained may serve to shed light on some physiological functions of these growth factors with respect to bovine oocyte maturation. MATERIALS
AND METHODS
In Vitro Oocyte Maturation Bovine ovaries were obtained from the slaughterhouse (GYPISA, Pozuelo de Alar&n, Madrid) and were transported in PBS (Dulbecco’s phosphate buffer saline) to the laboratory within 1 h. Follicular contents from small antral follicles (2 to 8 mm in diameter) were aspirated with a 18-gauge needle attached to a lo-ml disposable syringe. The oocytes were transferred onto a 35-mm plastic Petri dish (Bibby, Stone, Staffordshire, England, cat. no. 2333) containing 2 ml of Hepes-buffered washing medium (TCM 199 Hepes-buffered, Sigma Chemical Co. St. Louis, MO, cat. no. 2520) supplemented with 2% FCS (Gibco Ltd, Paisley, Scotland, cat. no. 6290-H), followed by 5 washes. According to predefined criteria (23), selected cumulus-oocytes were divided for culturing either as cumulus-oocyte complexes (with intact and unexpanded cumulus: COC) or denuded oocytes (without layers of cumulus cells), placed into drops (50 ~1) of maturation medium under oil and cultured on 35-mm Petri dishes at 39°C in 5% CO*, 95% air and 100% relative humidity for 24 h. The oocytes (5 per drop) were matured in TCM-199 (Earle’s salt with sodium bicarbonate and L-glutamine, Sigma, cat. no. 4530), this basic medium being supplemented with sera to form 3 treatment groups: Group I (without serum), Group II (with 10% v/v FCS), and Group III (with 10% v/v ECS). Each group then underwent 4 different, predefined treatments with growth factors (Boehringer Mannheim, Germany): control (no growth-factor), EGF (50 ng/ml), IGF-I (100 ng/ml), and EGF+IGF-I (50 and 100 ng/ml, respectively), for a total of 12 maturation treatments. Sera were heat-inactivated (56°C for 30 min) before use. The ECS was obtained from cows during standing estrus and was pooled as a stock reserved for use in this study.
111
Theriogenology Cumulus
Expansion
Evaluation
Cumulus oocyte complexes were evaluated at the end of the culture period in order to assess the effect of growth factors on cumulus expansion (Experiment 1). The cumulus expansion was scored based on the subjective scale of 0 to 3, in which 0 indicates no detectable expansion; + 1 indicates the minimum observable response (outer 1 or 2 layers expanded); t 2 indicates outer one-half of the cumulus expanded; and +3 indicates all layers of cumulus cells expanded, even those closest to the oocyte. Nuclear
Maturation
Evaluation
At the end of the culture period, all the oocytes were fixed to ascertain the influence of growth factors on nuclear maturation in vitro, and the effect of the presence or absence of cumulus cells surrounding the oocyte (Experiment 2). Cumulus cells of COCs were removed with hyaluronidase (200 UI/ml) and were mechanically stripped using a fine-bore pipette. The oocytes were then pipetted onto a slide. A coverslip spotted with a paraffinVaseline (1O:l) mixture at each corner was placed directly over the center of the drop containing the oocytes. Fixation of oocytes was carried out by placing the slides in acetic acid-ethanol (1:3) for 24 h and staining with aceto-orcein (2% orcein in 60% acetic acid) for 2 min. Nuclear maturation was evaluated and classified as germinal vesicle (GV), metaphase I (M I), metaphase II (M II) and degeneration. Statistical
Analysis
Other than the cumulus expansion experiment, which was performed 5 times, the nuclear maturation experiment was replicated 7 times. Statistical analysis using the Chisquare test was carried out in pooled data using the Biomedical Data Program (6); P values less than 0.05 were considered to be significant. Maximum cumulus expansion degree in Experiment 1 and Metaphase II stage in Experiment 2 were used as end-point parameters for assessing growth-factor and sera effect on bovine oocyte maturation. Data are present in results as percent mean r SEM of oocytes having undergone full expansion degree in Experiment 1 or M II stage in the Experiment 2. RESULTS Experiment
1
A total of 1105 bovine cumulus-oocyte complexes were used and evaluated in this experiment. Data for those COCs which attained full cumulus expansion are shown in Table 1. Compared with control and IGF-I treatments, EGF treatments resulted in a significantly higher incidence of cumulus expansion in Groups I and II (PcO.05) though not in Group III. When EGF was used in conjunction with IGF-I, cumulus expansion registered higher readings in all groups, these data being significantly higher than for treatments with IGF-I alone. No significant differences were observed between treatments with IGF-I and those without growth factors. In no case did COCs exhibit full expansion when cultured without growth factors and serum.
112
Theriogenology
Table 1. Cumulus expansion observed in bovine cumulus-oocyte complexes in response to various combinations of growth factors (EGF, 50ng/ml; IGF-I, 100 ng/ml; EGF+IGF-I, 50 and 100 ng/ml) and sera (none, FCS and ECS) after 24 hours of in vitro maturation Cumulus expansion % (observed/total oocytes) Treatment
Group I (Without serum)
Group II (10% FCS)
Group III (10% ECS)
TCM-199
0% (0/79)a
38.5% (32/83)a
44.8% (39/87)a
TCM-199+EGF
49.4% (45/91)h
62.6% (57/91)b
57.3% (51/89)a
TCM-199 + IGF-I
10.5% (10/95)a
40.6% (41/101)a
54.0% (53/98)a
TCM199 + EGF + IGF-I
55.3% (52/94)b
74.2% (69/93)bYc
75.9% (79/H@
Data were pooled from 5 independent experiments. Means are expressed (full expansion/total number of oocytes). Values with different superscripts significantly different: a vs b, PcO.05; a vs c, P< 0.01 by Chi-square test.
Experiment
as percentages in columns are
2
Oocyte totals, fixed and stained for maturity (n= 3386), are depicted in Figure 1 (COC) and Figure 2 (denuded oocytes). With regard to COCs, the highest metaphase II values in Group I (without serum) were recorded in response to EGF+IGF-I treatment (60.0 f 3.1%) and proved to be significantly different (PcO.05) from those recorded for IGF-I and control treatments. In Group II (with 10% FCS) the best maturation result was obtained with EGF+IGF-I (77.5 f 2.2%), there being significant differences compared with TCM199+FCS (41.6 f 4-l%, PcO.01) and IGF-I treatments (54.1 + 2.1%, PcO.05) but not with EGF treatment. Similarly, Group III (with 10% ECS) showed higher maturation means in response to EGF+ IGF-I treatment (75.8 k 2.3%), revealing statistical differences (P < 0.01) with TCM-199+ECS treatment. Nuclear maturation rates for treatments of all groups with serum were greater than for the group without. As between Groups II and III (both with serum), no differences were observed in treatments except when using IGF-I, which increased the maturation rate in the ECS-group compared with that of the FCS-group (64.5 f 2.3% vs 54.1 k 2.1%, PcO.05).
113
Theriogenology % of M II
**
**
80 70 60 50
n
NONE
EGF
IGF-I
(ill)
(148)
(113)
GROUP Figure 1.
No rate of the maturation treatments
I
EGF+IGF NONE (180)
(144)
EGF
IGF-I EGF+IGF NONE
EGF
IGF-1 EGF+IGF
(170)
(168)
(116)
(ZOO)
GROUP
III
GROUP
II
(156)
(141)
(207)
The effect of growth factors and sera on meiotic maturation of bovine cumulus-oocyte complexes. Oocytes were cultured in medium containing no added serum (Group I), 10% FCS (Group II), and 10% ECS (Group III) as well as with different growth factors (no growth factor; EGF, 50 ng/ml; IGF-I, 100 ng/ml, and EGF+IGF-I, 50 and 100 ng/ml). Metaphase II stage was assessed after 24 hours of culture. Data represent percentage means 2 SEM of 9 independent experiments. Bars with an asterisk indicate significant difference with the control (* P < 0.05; * * P < 0.01). significant differences among treatments were observed regarding the maturation denuded oocytes (P > 0.05, Figure 2). Cumulus-oocyte complexes exhibited higher values than denuded oocytes in all treatments, except for the control and IGF-I in Groups I and II.
DISCUSSION Immature oocyte development in the ovarian follicle to mature oocyte (metaphase II stage) depends on many regulatory factors (31,32). Cumulus oophorus expansion in bovine oocytes occurs in response to an ever-changing milieu of gonadotrophins, growth factors, steroids, factors secreted by the oocyte and other unknown molecules (3). These compounds could be contributing to maturational changes that occur in the oocyte, mediated by intracellular messengers such as CAMP, calmodulin or diacylglycerol (12). There are some criteria evaluating expansion of cumulus cells, and in the present study, we adopted similar expansion criteria to those used by other authors (3,8,29).
114
Theriogenology
% of M II 80 70 60 50
f
+ 40 30 20 10
-
-
0
n
NONE
EGF
IGF-I
EGF+IGF
NONE
EGF
IGF-I
(88)
wx
(171)
(144)
(103)
(148)
(114)
GROUP Figure 2.
I
GROUP
II
EGF+IGF (140)
NONE
EGF
IGF-I
(109)
(99)
(11’5)
GROUP
1 EGF+IGF (140)
III
The effect of growth factors and sera on meiotic maturation of bovine denuded oocytes. Oocytes were cultured in medium containing no added serum (Group I), 10% FCS (Group II), and 10% ECS (Group III), as well as with different growth factors (no growth factor; EGF, 50 ng/ml; IGF-I, 100 ng/ml, and EGF + IGF-I, 50 and 100 ng/ml). Metaphase II stage was assessed after 24 hours of culture. Data represent percentage means + SEM of 9 independent experiments. Bars with an asterisk indicate significant difference with the control. No significant differences with the control were observed.
Use was made of a serum-free group media (Group I) for in vitro maturation in order to explain the relationship between growth factors and the regulation of nuclear maturation and cumulus expansion, while effectively ruling out the influence of unknown serum factor (s). The results obtained indicate that EGF enhances cumulus expansion in bovine COCs in the same way as demonstrated by Downs (8) for murine oocytes. However, the lesser effect of EGF on bovine cumulus expansion compared to that seen in mouse oocytes may be due to the fact that, whereas recombinant human EGF was used in this study, homologous murine EGF was used in Downs’ mouse study (8). Although IGF-I per se is unable to promote effective cumulus expansion, this did not impair the maturation rate. Since IGF-I receptors have been detected in granulosa cells and oocytes (2), it is possible that suppression of expansion may be mediated by IGF-I acting on the granulosa cells directly or inhibiting the production of an expansion factor produced by the oocyte itself (3).
Theriogenology
115
Other authors using IGF-I (16) or IGF-I analogues, such as insulin (36), likewise recorded poor results for bovine cumulus expansion. The greater effect exerted by growth factors plus serum (FCS or ECS) on bovine cumulus expansion can be explained on the basis of the hormonal content of such sera. Both FCS and ECS are routinely used in culture media for oocyte maturation (10,11,22,27), yet they differ in the comparatively greater concentrations of LH, TSH and estradiol-170 present in ECS (33,34). Addition of sera to growth-factor media has an effect on EGF and EGF+IGF-I induced cumulus expansion, suggesting that these compounds may be acting in the cumulus cells through a synergic mechanism. In the present study, both EGF and EGF+IGF-I combined, with or without serum, effectively stimulated cumulus expansion. To date, however, there has been no study describing the interactions between these 2 growth factors with the aim of evaluating cumulus expansion. The highest percentages of EGF-induced nuclear maturation correspond to those obtained in rodent oocytes (58). Such high rates can be attributed to the fact that the main agent involved in the inhibition of meiotic resumption in these species’ oocytes is the stimulation of CAMP-dependent protein kinase. However, in their response to protein kinase stimulation via CAMP accumulation, bovine oocytes seem to differ from those of the above species (30), possessing other substances inhibiting meiotic activation (e.g., purines) upon which EGF, in all likelihood, fails to act in the same way. In our maturation experiment, the highest results were achieved with the addition of EGF+IGF-I, such data being in line with the recent results obtained in porcine oocytes (25). This could indicate that, under in vivo conditions, there may be an additive element at work in the way in which these two growth factors combine their respective actions. They bring about specific actions on oocyte maturation, including cumulus and cytoplasmic effects. Hainaut et al. (13) postulated that maturation with IGF-I is initiated upon activation of the membrane receptor for this growth factor and requires tyrosine dephosphorylation of ~34, the kinase component of maturation promoting factor (MPF). Otherwise, the IGF-I in the above cited reports, did not stimulate GVBD of mouse oocytes but did stimulate the resumption of meiosis for rat oocytes. In the present study, IGF-I, when acting singly, had a slight stimulatory effect on bovine-oocyte nuclear maturation. As between groups with sera, no significant differences in the case of maturation treatments were observed, except in the case of treatments using IGF-I alone; here, the maturation percentage was higher in the ECS- than in the FCS-group. IGF-I increases the number of receptors for LH in granulosa cells (1); hence, by using ECS, which has a higher LH concentration than FCS, nuclear maturation is boosted. These results were only observed in cumulus-oocyte complexes since LH needs cumulus cells if it is to act effectively on the oocyte (35). The obtained M II rates in COCs were low compared with those of other reports; these may have been due to variations in the FCS-commercial lots (10,28) or to the hormonal concentration of ECS (33). Ovarian and intrafollicular mechanisms for the regulation of oocyte growth and maturation are complex. The mechanisms whereby growth factors regulate or modulate resumption of meiosis in oocytes may be mediated via the granulosa and/or cumulus cells (4,5). It has been previously demonstrated that the growth growth factors can be synthesized by the ovary (14,24). The presence of EGF or EGF-like substances has been demonstrated for porcine and human follicles at concentrations similar to those used by us (19,21).
116
Theriogenology
Moreover, concentrations of IGF-I, similar to those used in the present study, have been detected in bovine and porcine follicular fluid (15). The growth factors and/or serum used in this study enhanced maturation in cumulus-oocyte complexes but not in denuded oocytes. Not only is this in accordance with the results observed in rodents (5,8) and those obtained using other growth factors such TGF-a (4), but it also demonstrates that growth-factor action requires the presence of cumulus cells, by which a positive stimulus for nuclear maturation is transferred to the oocyte. This paper provides evidence: 1) that inducer of meiotic maturation and cumulus presence of FCS or ECS enhances the effect and meiotic maturation in immature bovine
EGF, singly or combined with IGF, is a potent expansion in bovine oocytes; and 2) that the of these growth factors on cumulus expansion cumulus-oocyte complexes.
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12. Goncalves PB, Graves CN. Interaction between CAMP, diacylglycerol and Ca”calmodulin pathways in the regulation of cumulus expansion. Biol Reprod 1992; 46 (Suppl. 1):69 abstr. 13. Hainaut P, Giorgetti S, Kowlaski A, Ballotti R, Van Obberghen E. Antibodies to phosphotyrosine injected to xenopus laevis oocytes modulate maturation induced by insulin/IGF-I. Exp Cell Res 1991; 195129-136. 14. Hammond JM, Baranao JLS, Skaleris D, Knight AB, Romanus JA, Rechler MM. Production of insulin-like growth factors by ovarian granulosa cell. Endocrinology 1985; 117:2553-2555. 15. Hammond JM, Hsu CJ, Klindt J, Tsang BK, Downey BR. Gonadotropins increase concentrations of immunoreactive insulin-like growth factor-I in porcine follicular fluid in vivo. Biol Reprod 1988; 38:304-308. 16. Herrler A, Lucas-Hann A, Niemann A. Effects of insulin-like growth factor-I on in vitro production of bovine embryos. Theriogenology 1992; 37:1213-1224. 17. Hernandez ER, Resnick CE, Svoboda ME, Van Wyk JJ, Payne DW, Adashi EY. Somatomedin-C/insulin-like growth factor I as an enhancer of androgen biosynthesis by cultured rat ovarian cells. Endocrinology 1988; 122:1603-1612. 18. Hillensjo T, Ekholm C, Ahren K. Role of cylic AMP in oocyte maturation and glycolysis in the pre-ovulatory rat follicle. Endocrinology 1978; 122:377-388. 19. Hoffmann GE, Scott RT Jr, Brzyski RG, Jones HW Jr. Immunoreactive epidermal growth factor concentration in follicular fluid obtained from in vitro fertilization. Fertil Steril 1990; 54:303-307. 20. Hsu C, Hammond JM. Gonadotropins and estradiol stimulate immunoreactive insulinlike growth factor-I production by porcine cells in vitro. Endocrinology 1987; 120:198207. 21. Hsu CJ, Holmes SD, Hammond JM. Ovarian epidermal growth factor-like activity: concentrations in porcine follicular fluid during follicular enlargement. Biochem Biophys Res Commun 1987; 147( 1):242-247 22. Leibfried-Rutledge ML, Critser ES, First NL. Effect of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulusoocyte complexes. Biol Reprod 1986; 35:850-857. 23. Lorenzo P, Illera MJ, Illera JC, Illera, M. Acciones especificas de 10s factores de crecimiento (EGF e IGF-I) en la maduracion in vitro de oocitos bovinos. Rev Esp Fisiol 1993; 49:265-270. 24. Makris A, Klagsbrum MA, Yasaumizu T, Ryan KJ. An endogenous ovarian growth factor with stimulates BALB/3T3 and granulosa cell proliferation. Biol Reprod 1983; 29:1135-1141. 25. Reed ML, Estrada JL, Illera MJ, Petters RM. Effects of epidermal growth factor, insulin-like growth factor-I, and dialyzed porcine follicular fluid on porcine oocyte nuclear maturation in vitro. J Exp Zoo1 1993; 266:74-78. 26. Sanbuissho A, Threlfall WR. The effect of estrous cow serum on the maturation and fertilization of the bovine follicular oocyte in vitro. Theriogenology 1989; 31:693-699. 27. Schellander K, Fuhrer G, Brackett BG, Korb H, Schleger W. In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum. Theriogenology 1990; 33:477-485. 28. Shiigi SM, Mishell RI. Sera and the in vitro induction of inmune responses. I. Bacterial contamination and generation of good fetal bovine sera. J Inmunol 1975; 115:741 abstr.
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29. Singh B, Barbe CJ, Armstrong DT. Factors influencing resumption of meiotic maturation and cumulus expansion of porcine oocyte-cumulus cell complexes in vitro. Mol Reprod Dev 1993; 36:113-119. 30. Sirard MA, Coenen K, Bilodeau S. Effects of fresh or cultured follicular fractions on meiotic resumption in bovine oocytes. Theriogenology 1992; 37:39-58. 31. Tonetta SA, DiZerega GS. Intragonadal regulation of follicular maturation, Endocrine Rev 1989; l&205-229. 32. Wassarman PM. The mammalian ovum. In: Knobil E, Neil1 JD (eds), The Physiology of Reproduction. New York: Raven Press, 1988;69-102. 33. Younis AI, Brackett BG, Fayrer-Hosken RA. Influence of serum and hormone on bovine oocyte maturation and fertilization in vitro. Gamete Res 1989; 23:189-201. 34. Younis AI, Brackett BG. Thyroid stimulating hormone enhancement of bovine oocyte maturation in vitro. Mol Reprod Dev 1992; 31:144-151. 35. Zuelke KA, Brackett BG. Effect of luteinizing hormone on glucose metabolism in cumulus enclosed bovine oocytes matured in vitro. Biol Reprod 1992; 46(Suppl. 1):117 abstr. 36. Zhang L, Blakewood EG, Denninston RS, Godke RA. The effect of insulin on maturation and development of in vitro-fertilized bovine oocytes. Theriogenology 1991; 35:301 abstr.