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Regular Article |
a Department of Population Health & Reproduction, School of Veterinary Medicine, University of California, Davis, California 95616
b Animal Physiology Department, Veterinary School, Universidad Complutense de Madrid, 28040 Madrid, Spain
c Department of Animal Reproduction, Faculdade de Veterinaria, Universidade Federal de Pelotas, RS 96010-900, Brazil
d Animal Reproduction Laboratory, Temperate Climate Research Corporation, EMBRAPA, BR 392 Km 78 Cx. P. 403 Pelotas, RS 96001-970, Brazil
e Department of Animal Science, University of California, Davis, California 95616
ABSTRACT
The effects of insulin-like growth factor-I (IGF-I) and its interaction with gonadotropins, estradiol, and fetal calf serum (FCS) on in vitro maturation (IVM) of equine oocytes were investigated in this study. We also examined the role of IGF-I in the presence or absence of gonadotropins, estradiol, and FCS in parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), using cleavage rate as a measure of cytoplasmic maturation. Only equine cumulus-oocyte complexes with compact cumulus and homogenous ooplasm (n = 817) were used. In experiment 1, oocytes were cultured in TCM-199 supplemented with BSA, antibiotics, and IGF-I at 0 (control), 50, 100, 200 ng/ml, at 39°C in air with 5% CO2, 95% humidity for 36 or 48 h. In experiment 2, oocytes were cultured with FSH, LH, estradiol, and FCS with IGF-I at the concentration that promoted the highest nuclear maturation rate in experiment 1. In experiment 3, oocytes from the three experimental groups (IGF-I; hormones; and IGF-I + hormones) were chemically activated by exposure to calcium ionophore followed by culture in 6-DMAP. In experiment 1, IGF-I stimulated equine oocyte maturation in a dose-dependent manner with the highest nuclear maturation rate at a concentration of 200 ng/ml. No significant effect of IGF-I on nuclear maturation was observed in experiment 2. In experiment 3, a significant difference in cleavage rate was observed between the hormone + IGF-I group (15 of 33; 45.4%) compared with IGF-I (10 of 36; 27.8%) and hormone (4 of 31; 12.9%) alone (P < 0.05). These results demonstrated that IGF-I has a positive effect on nuclear maturation rate of equine oocytes in vitro. The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation of equine oocytes measured by parthenogenic cleavage.
developmental biology, early development, growth factors, insulin-like growth factor receptor, oocyte development
INTRODUCTION
In vitro fertilization (IVF) has been routinely used in humans and several domestic animal species; however, limited success has been achieved in horses. Based on several reports, immature equine oocytes are capable of completing meiosis in vitro, but subsequent fertilization and embryonic development of those oocytes are questionable [1]. This suggests that the problems encountered in in vitro production of fertilizable eggs in the horse are due at least in part to inadequate in vitro maturation (IVM) procedures. Several variations of medium and incubation periods have been evaluated with limited improvement [1–4]. In most of these studies, the endpoint was nuclear maturation rate recorded as percentage of oocytes that achieved metaphase II (M-II). The ability of fertilized oocytes to develop depends upon normal nuclear and cytoplasmic oocyte maturation [5]. Both are essential for an oocyte to develop the capacity for fertilization and normal embryonic development. In the horse, the study of oocyte cytoplasmic maturation and postfertilization development has been hindered by the lack of progress in IVF.
In mammalian oocytes, parthenogenesis and cellular division require cytoplasmic maturation with assembly of major cytoskeletal components and reorganization of organelles [6]. The integral distribution of microtubules and microfilaments has been demonstrated as a need for their assembly for pronuclear formation and migration, as well as cleavage after fertilization and parthenogenesis [6]. Gonadotropins are the main regulators of nuclear maturation of oocytes; however, recent observations suggest that gonadotropins are only part of a complex system of autocrine and paracrine factors that may influence maturation. Growth factors have a regulatory role in ovarian function [7]. Among them, insulin-like growth factor-I (IGF-I) has been implicated in the regulation of follicular somatic cell functions, including differentiation, proliferation, and steroidogenesis [8, 9]. Insulin-like growth factor-I is a potent mitogen for granulosa cells [10], even in the absence of FSH [11], and acts as a biological amplifier of the action of FSH in the ovary [12]. Also, studies performed in mice with a complete deficiency of IGF-I (IGF-I-null mice), created by homologous recombination, found that these animals exhibited infertility associated with a reduced level of granulosa FSH receptor expression, reduced aromatase expression, and failed to develop normal follicles beyond the early antral stage. This study suggested also that ovarian IGF-I expression enhance granulosa cell FSH responsiveness by augmenting FSH receptor expression [13].
The regulation of oocyte maturation by growth factors in animal species such as mice [14], rats [15], rabbits [16], pigs [17, 18], and cattle [19, 20] has been described. Moreover, immunoreactive IGF-I has been localized in the theca-interstitial compartment and in the cumulus cells surrounding the human oocyte [21]. Insulin-like growth factor-I stimulates maturation in Xenopus oocytes [22], enhances bovine oocyte maturation and fertilization in vitro [23, 24], and promotes rabbit blastocyst development [25], suggesting an important role not only in the nuclear maturation of oocytes but also in cytoplasmic maturation. In these studies, nuclear and cytoplasmic maturation were measured by IVF and blastocyst formation.
In vivo maturation conditions in the mare differ from those of other domestic mammals. Ovarian physiology of the mare is an interesting model displaying some unique features: a long period of estrus, a large preovulatory follicle, and a progressive increase in LH lasting many days, with peaks observed 1 day after ovulation [26, 27]. The optimal hormonal pattern for follicular growth and development is not well known and is still being investigated in the horse. It is clear that FSH and estradiol (E2) are important components for IVM [1–4]. In addition, one might assume that equine oocytes possibly require the influence of LH for several days to mature. A close relationship between IGF-I, hormones and oocyte maturation is possible. To gain insight into this process, we evaluated the effect of IGF-I and its interaction with FSH, LH, E2, and fetal calf serum (FCS) on IVM of equine oocytes during culture for 36 or 48 h. We also examined the role of IGF-I in the presence or absence of those components on parthenogenic cleavage after oocyte activation with calcium ionophore combined with 6-dimethylaminopurine (6-DMAP), with cleavage rate as the measure of cytoplasmic maturation. The addition of calcium ionophore along with 6-DMAP has been shown to be particularly effective in inducing bovine oocyte activation [28]. Our hypothesis was that nuclear and cytoplasmic maturation of equine oocytes is enhanced by the addition of IGF-I to the culture medium.
MATERIALS AND METHODS
A total of 1002 ovaries were recovered with 1449 oocytes retrieved (2.89 oocytes/mare). After selection 817 oocytes were used in the experiments.
Collection of Cumulus-Oocyte Complexes (COCs)
Ovaries were collected from a slaughterhouse located 20 min from the Research Center in Pelotas RS Southern of Brazil (parallel 32°S). Ovaries were placed in PBS containing 1000 IU/ml of penicillin, and 500 µg/ml of streptomycin within 3 h of slaughter and were transported to the laboratory in a thermal container at 25–30°C. The interval from slaughter to arrival at the laboratory ranged from 20 min to 3 h. Before oocyte collection ovaries were dissected, and the tunica albuginea was removed allowing visualization of follicles on the ovarian surface. The ovaries were washed three times in PBS and antibiotics. Follicular contents were aspirated through an 18-gauge needle connected to a 35-ml syringe. During aspiration, a scraping motion within the follicle was done with the needle after the first aspiration, and the follicle was washed with its own follicular fluid. After 20 min of sedimentation, the pellet was collected, placed in 100-mm Petri dishes and examined with a stereomicroscope to locate the COCs. After all visible follicles had been punctured, ovaries were cut into slices of about 2 mm with a razor blade to find other follicles within the ovarian stroma. Oocytes were collected from these follicles by scraping with a bone curette. The follicular contents collected in this procedure were placed into 100-mm Petri dishes. Oocytes were collected and placed in a drop of follicular fluid and examined for morphology. Oocytes selected for culture were washed three times. The basal medium for oocyte washing and maturation was tissue culture medium-199 (TCM-199) with 0.1% BSA (A-3311 fraction V; Sigma Chemical Co., St. Louis, MO), 100 IU/ml of penicillin, and 50 µg/ml of streptomycin. The interval between collection of oocytes from follicles and culture was less than 1 h, and the overall interval between obtaining ovaries from the slaughterhouse and culture of oocytes ranged from 1.5 to 5 h.
Culture of COCs
Insulin-like growth factor-I stock solution (I-3769; Sigma) was diluted in TCM-199 and added to the maturation medium in different concentrations. Only oocytes with compact cumulus and homogenous cytoplasm (n = 817) were used. In experiment 1 (n = 344), COCs retained for maturation were randomly allocated to two different culture periods of 36 and 48 h. Cumulus-oocyte complexes were cultured in a 4-well Petri dish (no. 176740; Nunc Intermed., Roskilde, Denmark) containing 500 µl of the basal medium supplemented with IGF-I at 0 (control), 50, 100, 200 ng/ml, and cultured at 39°C in air with 5% CO2, 95% humidity. Neither serum nor hormones were added to the maturation medium. These concentrations were chosen based on follicular fluid levels reported in earlier studies in humans [29], ovine [30], and bovine [31]. At the end of the 36- or 48-h culture period, cumulus cells were removed with 0.1% hyaluronidase solution (H-3506, Sigma) and mechanically stripped. Oocytes were stained with 10 µg/ml of bis-benzamide (Hoechst 33342; Sigma B-2261) for 20 min at 39°C, and pipetted onto a slide containing a strip of silicone sealant (Silicone 732; Saf-t-lok Chemical Corp., Lombardi, IL) at the top and the bottom. A coverslip was placed directly over the center of the drop containing the oocytes within a 25-µl drop of mounting medium (H-1300; Vector Labs., Inc., Burlingame, CA). The oocytes were evaluated using an epifluorescence microscope with a 365-nm exciter filter and Chroma 81P101 Pinkel #1 emission filter to evaluate nuclear maturation. Nuclear maturation was recorded as percentage of oocytes that had reached M-II by the presence of the chromosomes in the metaphase plate and presence of the first polar body (Fig. 1). Data from 13 replicates were pooled.
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In experiment 2 (n = 179), oocytes were selected as in experiment 1, randomly allocated to two different culture periods of 36 and 48 h in TCM-199 with antibiotics containing 5 IU/ml porcine FSH (Sioux Biochemical), 10 IU/ml equine LH (L9773; Sigma), 1 µg/ml E2 (E-8875; Sigma), 10% heat-inactivated FCS (F-3018; Sigma) [32, 33] in the presence and absence of IGF-I with the concentration that promoted the highest nuclear maturation rate in experiment 1. These groups were referred to as hormone and hormone + IGF-I. Data from nine replicates were pooled.
In experiment 3 (n = 294), oocytes from the three experimental groups (IGF-I, hormone, and hormone + IGF-I), cultured for 36 and 48 h, were chemically activated by exposure to 10 µM ionomycin (I-0634; Sigma) for 5 min, washed in Tyrode albumin lactate pyruvate (TALP)-Hepes medium, followed by culture in 1.9 mM 6-DMAP (D-2629; Sigma), a puromycin analog and phosphorylation inhibitor, for 3 h [28]. After activation, oocytes were placed in culture in TCM-199 supplemented with 10% FCS and evaluated for parthenogenic cleavage every 24 h until nuclear division stopped or for a maximum period of 8 days. The control group for the activation experiment included placing oocytes matured in the three groups for 5 min in Hepes-buffered Tyrode solution (HbT-vehicle for ionomycin and 6-DMAP), washed in TALP-Hepes medium, and placed for 3 h in HbT. Oocytes were later transferred to TCM-199 supplemented with FCS following the same protocol as with the chemical activators. Parthenotes were stained with 10 µg/ml bis-benzamide and evaluated for the presence of nuclei using an epifluorescence microscope. Data from nine replicates were pooled.
Data Analysis
Analysis of the effects of IGF-I on IVM was performed by logistical regression analysis. The differences in oocyte maturation after different IVM treatments and IVM culture periods were analyzed by chi-square test. The proportion of oocytes activated compared with controls, and the comparison of activated oocytes from different IVM groups was also analyzed by chi-square test. The level of significance was set at P < 0.05.
RESULTS
A total of 1449 oocytes was retrieved. Strict selection of oocytes was performed based on morphologic criteria, and only 817 oocytes (56%) were used to evaluate treatment effects.
Nuclear Maturation
In experiment 1 (n = 344), IGF-I stimulated oocyte maturation in a dose-dependent manner, with the largest effect observed at a concentration of 200 ng/ml (60%) compared with oocytes cultured in the absence of IGF-I (32%) during 36 h of culture (Table 1). A significant (P = 0.003), positive linear trend was observed in the log odds of nuclear maturation with increasing IGF-I concentration (ng/ml) after 36 h. No significant effect was observed after 48 h of maturation with IGF-I; however, a numeric difference (P = 0.1) was seen between controls within the 36- versus 48-h culture periods (32.1% versus 48.4%). In experiment 2 (n = 179), COCs were matured for 36 or 48 h with gonadotropins and FCS in the presence or absence of 200 ng/ml IGF-I that elicited the largest meiotic response of oocytes in experiment 1. No significant effect of IGF-I on nuclear maturation was observed during either 36 or 48 h of culture. Results are summarized in Table 2.
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Cytoplasmic Maturation
The role of IGF-I in cytoplasmic maturation in the presence or absence of FCS and hormones was examined to measure parthenogenic cleavage after oocyte activation as an indirect measure of cytoplasmic maturation. The overall cleavage rate was 28.5% (57 of 200), and there was no significant difference in the cleavage rate at 36 or 48 h of culture period. For both maturation periods, a higher cleavage rate was seen in the group containing 200 ng/ml IGF-I in addition to FCS and hormones (P < 0.05) compared with IGF-I and hormone groups alone. Spontaneous activation was measured in the control group. A highly significant difference was observed between the group containing 200 ng/ml IGF-I, FCS, and hormones compared with controls (P = 0.0007). From a total of 57 cleaved oocytes (Fig. 2), 5 (8.8%) reached the morula stage (Fig. 3). The control group showed a rate of spontaneous cleavage of 3.2% (3 of 94). Results are summarized in Table 3. The total cleavage rate is shown in Table 4.
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DISCUSSION
The results of this study showed that IGF-I has a positive effect on equine oocyte in vitro nuclear maturation rate in the absence of FCS. They also showed that IGF-I stimulates oocyte maturation in a dose-dependent manner during a 36-h period, with the largest effect seen at a concentration of 200 ng/ml (60% versus 32%). The addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation, but resulted in a positive effect on cytoplasmic maturation measured by parthenogenic cleavage, suggesting an interaction between factors that act synergistically in nuclear and cytoplasmic maturation of equine oocytes.
These findings are consistent with those of previous reports that IGF-I enhanced the nuclear maturation of oocytes in the rat [15], human [34], and rabbit [16, 35]. In contrast, IGF-I did not appear to influence nuclear maturation in the mouse [36], pig [17], and cow [20]. The results also provided further insight on the potential interaction of gonadotropins with IGF-I influencing cytoplasmic maturation as evidenced by a higher parthenogenic cleavage rate. These findings are in agreement with results obtained in water buffalo [37] indicating that IGF-I induced proliferation of granulosa cells and maturation of oocytes, increasing cleavage and blastocyst development rates when the oocytes were matured in the presence of IGF-I. Results also agree with those in the rabbit, which demonstrated that IGF-I supports early embryonic development by preventing apoptosis and by increasing cell proliferation [25]. No difference in nuclear maturation rate was observed among treatments during 48-h culture. However, a numeric difference (P = 0.1) was seen between the control groups in the absence of IGF-I during 36 and 48 h of maturation (32.1% in 36 h versus 48.4% in 48 h). This difference could suggest that in the absence of IGF-I, horse oocytes require a longer IVM period in order to reach M-II. Moreover, in the control group and after 36 h of maturation, the cumulus cells were resistant to mechanical removal from the oocytes compared with those incubated for 48 h. Additionally, a higher percentage rate of oocytes in the 36-h culture group remained in metaphase-I, suggesting that the oocytes likely could have reached M-II after a longer culture period. The cumulus cells surrounding the oocytes in the control group were easily removed after 48 h of IVM, suggesting complete oocyte maturation by disruption of the communication between the oocytes and cumulus cells [38].
In experiment 1 serum-free medium was used for IVM to establish the effect of IGF-I without possible influence by unknown factor(s) present in the serum. Concentrations of IGF-I similar to those used in these experiments have been reported in humans [29], ovine [30], porcine [39], and bovine [31, 40] follicular fluid, suggesting a possible role of this growth factor in the regulation of nuclear maturation in vivo. No statistical differences were observed in nuclear maturation rate between the TCM + 200 ng/ml IGF-I group and the group containing gonadotropins, E2, and FCS (60% versus 63%). To determine if the addition of IGF-I to the hormone group would increase maturation rate, we supplemented the medium containing gonadotropins and FCS with 200 ng/ml IGF-I. No difference in nuclear maturation rate was observed. In blood or in other body fluids, IGF-I and IGF-II are associated with specific IGF-binding proteins (IGFBPs) that appear to regulate the physiologic actions of IGFs, in part by modulating their interactions with their specific cell-surface receptors [41–43]. Six high-affinity IGFBPs have been fully characterized, with a further four proposed by researchers. Moreover, an emerging group of low-affinity IGFBPs has also been characterized. Several investigators have hypothesized that IGFBPs have a regulatory role in vivo, modulating the biological activity of IGF-I. Thus, the FCS utilized in this experiment might have contained IGFBPs that regulate the action of IGF-I in an autocrine/paracrine fashion at the cellular level. It has been reported that these IGFBPs either increase [41] or decrease [42, 43] the response of cells to IGFs. Another explanation is that IGF-I could have been present in the FCS in a concentration that reached the peak of biological activity and when supplemented with 200 ng/ml of IGF-I, it did not have significant effect.
Oocyte activation is associated with a calcium influx during fertilization [44]. Activation can be achieved artificially in the absence of fertilization by mimicking the calcium influx via electrical pulse [45], use of chemicals such as calcium ionophore [44, 46], ethanol [46, 47], hyaluronidase [46, 48], and lowered temperature [49]. In the cow and rabbit, oocytes activated parthenogenetically can develop to the blastocyst stage [47, 50]. Oocytes matured for a period longer than that required to reach M-II (aged oocytes) activate more readily than younger oocytes [51, 52]. In a preliminary experiment (data not shown), we matured oocytes at different time periods to test this hypothesis and did not observe differences in activation between our controls and treatment after 36 and 48 h. Oocytes matured for more than 48 h did not respond to the activation treatment, indicating loss of viability or death. After maturation for 48, 60, and 72 h, oocytes were stained with Hoechst, and the 48-h control group in the absence of IGF-I showed a higher M-II rate compared with the 36-h group. These preliminary results encouraged the inclusion of a 48-h IVM group in our study. In vivo maturation of equine oocyte is accompanied by a progressive increase in LH concentration lasting for many days, with peaks being observed 1 day after ovulation. Moreover, in vivo response to hCG injections in mares containing follicles >35 mm requires 30–48 h to induce ovulation, and one could speculate that equine oocytes need a longer culture period to achieve complete cytoplasmic maturation. However, further studies are required because our results showed no statistical differences in cleavage rate between incubation periods of 36 and 48 h.
Ideally, acquisition of developmental competence as demonstrated by IVF and early embryonic development is a more reliable criterion than parthenogenic cleavage, but the lack of an ideal equine IVF system prevents us from using oocyte developmental competence as an endpoint. Several investigators have demonstrated the occurrence of a series of cytoskeletal events during parthenogenesis that are very similar to that of fertilization, such as microtubule and microfilament assembly, DNA synthesis, cortical granule migration and exocytosis, the presence of extensive meiotic and mitotic figures, nuclear organization with intact envelopes, and cellular division [6, 53–55]. Results from all of these studies suggest that cytoplasmic maturation is necessary as a prerequisite in order for such dynamic events to occur.
The cleavage rate observed in this study suggests an IVM protocol that may serve as indirect evidence for both nuclear and cytoplasmic maturation in the horse. The study of parthenogenesis is useful as an approach to understanding the fundamental aspects of early embryonic development. In this context, parthenogenic development of an unfertilized oocyte to the morula stage, as was demonstrated in this study, could be a useful model to study embryogenic competence in the horse without including the confounding effects of sperm and fertilization treatments. To our knowledge, no reports are available on in the vitro development to the morula stage of parthenogenetically activated equine oocytes after IVM. An important factor in this study could have been the strict selection of oocytes (from all retrieved oocytes, fewer than half were selected for allocation to treatments based on their morphology). The capability of equine oocytes to be activated has been described [46], but no cleavage was reported.
In conclusion, results of this study showed that IGF-I has a positive effect on the in vitro nuclear maturation rate of equine oocytes during 36 h of culture. We also demonstrated that equine oocytes can develop parthenogenetically to the morula stage. Moreover, results of our study demonstrate that the addition of IGF-I to an IVM medium containing hormones and FCS did not increase nuclear maturation but promoted cytoplasmic maturation as measured by parthenogenic cleavage. This observation suggests a synergistic interaction between IGF-I, gonadotropins, E2, and FCS in nuclear and cytoplasmic maturation of equine oocytes. Because an effective IVF protocol has not been resolved for use in the horse, understanding parthenogenic activation is relevant and a basic concern in embryo cloning research because artificial activation of oocytes is an essential component of the current nuclear transfer protocols.
ACKNOWLEDGMENTS
The authors acknowledge the excellent technical assistance from Anita Mylius Pimentel, Marcia Duarte, Marcelo Mendonça, Dr. Gabriel Pereira, and Dr. Anelis Coscioni, as well as Dr. Philip Kass and Dr. Paulo Duarte for statistical advice.
FOOTNOTES
First decision: 7 February 2001.
1 This project was supported in part by Club Hipico La Silla, Monterey, Mexico, and by Center for Equine Health with funds provided by the Oak Tree Racing Association, the State of California parimutuel fund, and contributions by private donors. G.C. is a research fellow recipient of CAPES/MEC, Brazil. Part of this work was presented as a poster at the International Embryo Transfer Society Meeting (IETS), Omaha, NE, 13–16 January 2001. 
2 Correspondence: Irwin K.M. Liu and Gustavo F. Carneiro, Department of
Population Health and Reproduction, School of Veterinary Medicine,
1114 Tupper Hall, University of California, Davis, CA 95616. FAX: 530
752 4278; ikliu{at}ucdavis.edu and gfcarneiro{at}ucdavis.edu
Accepted: April 30, 2001.
Received: January 17, 2001.
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