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Origin of bovine follicular fluid and its effect during in vitro maturation on the developmental competence of bovine oocytes
Theriogenology 62 (2004) 1596–1606
Origin of bovine follicular fluid and its effect during in vitro maturation on the developmental competence of bovine oocytes Atef Alia, Karine Coenena, Daniel Bousquetb, Marc-Andre´ Sirarda,* a
Department of Animal Science, Centre de Recherche en Biologie de la Reproduction (CRBR), Laval University, Ste-Foy, Que., Canada G1K 7P4 b L’Alliance Boviteq Inc., St-Hyacinthe, Que., Canada J2S 7A9 Received 7 July 2003; accepted 3 March 2004
Abstract Protein supplementation during in vitro maturation can profoundly affect both the rate and overall efficiency of the maturation procedure [Biol. Reprod. 66 (2002) 901]. The present study was conducted to assess the ability of different concentrations (1, 5, and 10%) of bovine follicular fluid (bFF) to support in vitro maturation of oocytes and subsequent developmental capacity. The bFF was derived either from competent follicles (>8 mm) obtained by transvaginal recovery following superovulation or from a pool of small follicles (2–5 mm) from abbatoir-derived ovaries. Bovine oocytes were cultured for 24 h in synthetic oviduct fluid medium (m-SOF) supplemented with polyvinylpyrrolidone. Following fertilization and embryo culture, more oocytes (P < 0:05) reached the blastocyst stage when oocytes were cultured with 5% bFF from competent follicles (41 3:7%) compared with bFF derived from small follicles (16 2:9%). Estradiol and recombinant human follicle stimulating hormone added to the competent bFF during maturation acted in synergy to increase blastocyst production rate (P < 0:05); this blastocyst production rate (57 1:2%) was higher than those obtained with the addition of these two hormones to bFF derived from small follicles (26 2:9%). The quality of blastocysts obtained was reflected by inner cell mass (51:30 3:5 and 25:50 3:7) and trophectoderm cell numbers (99:72 2:5 and 94:80 4:7) for bFF from competent and small follicles, respectively. In conclusion, follicular fluid originating from competent follicles increased the developmental competence of abbatoir-derived oocytes. # 2004 Elsevier Inc. All rights reserved. Keywords: Bovine oocyte; Follicle; Early development; In vitro fertilization; Embryo
* Corresponding author. Fax: þ1 418 656 3766. E-mail address: [email protected] (M.-A. Sirard).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.03.011
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1. Introduction When comparing oocytes matured in vivo versus in vitro, no apparent differences are evident in the level of nuclear maturation, in the rates of in vitro fertilization or cleavage, but rather in the in vitro developmental competence of the oocytes [2,3]. Better cytoplasmic maturation of oocytes matured in vivo could explain the higher developmental competence observed [2]. Bovine oocytes resuming meiosis in vivo originate from dominant follicles about 15 mm that have been grown from 4 to 15 mm in approximately 5 days [4]. In contrast, oocytes for in vitro maturation (IVM) are usually retrieved from 2 to 6 mm follicles that are between 4 and 10 days from potential ovulation and the IVM period lasts only 24 h [5]. Thus, it is possible that oocytes matured in vitro have not acquired maximal developmental competence due to incomplete oocyte capacitation, a process during which RNA, proteins, and other molecules are synthesized [6]. Intrinsic factors such as the size of the follicle of origin could influence the percentage of bovine embryos produced in vitro [5,7,8]. Larger follicles contain oocytes more likely to develop into blastocysts than oocytes from smaller follicles. How the ovaries were handled before the culture is also an important factor that can affect the expression of developmental competence. Blondin et al. [9] found that bovine oocytes derived from ovaries maintained at 35 8C for 4 h after slaughter yielded higher frequencies of blastocyst development than those collected 2, 6 or 7 h post-slaughter, thus indicating that some developmental competence may be acquired shortly prior to in vitro maturation. Our laboratory has also demonstrated that the ‘‘coasting’’ period between hormonal stimulation and ovary collection [10] as well as the time interval from ovary collection to oocyte aspiration [9] significantly affected the developmental potential of COC. Addition of selected bovine follicular fluid (bFF) to the maturation medium influenced the developmental potential of oocytes obtained from unselected ovaries [11–14]. The use of bFF as a supplement to the maturation medium generally resulted in improved cytoplasmic maturation (to an extent similar to that achieved with fetal calf serum). The effect of bFF is not always affected by follicle size but may be affected by the quality offollicle from which it is obtained [12,14]. Exposure of oocytes to bFF collected at different times after LH surge profoundly affects rates of blastocyst formation [13]. In fact, the beneficial effect on development was present in dominant follicles, but not in all growing or regressing dominant follicles [11], suggesting subtle differences between growth and differentiation signals. It appears that follicular fluid influences oocyte competence, developmental capacity, and embryonic quality. To test this hypothesis, COCs were incubated in completely defined maturation medium supplemented with various concentrations of bFF derived from competent follicles (>8 mm) or small follicles (2–5 mm), or bovine serum albumin fraction V (BSA-V; as a source of nonspecific protein). 2. Materials and methods 2.1. Oocyte recovery and selection Bovine ovaries at various stages of their estrous cycle were obtained from a abbatoir and transported to the laboratory in a 0.9% NaCl aqueous solution containing 100,000 IU/L
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penicillin, 100 mg/L streptomycin, and 250 mg/L amphotericin B (Sigma-Aldrich, Oakville, Ont., Canada). Cumulus-oocyte complexes (COCs) were aspirated from 2 to 6 mm follicles with an 18-g needle attached to a 10-mL syringe. Only oocytes with 4 layers of compact cumulus cells and homogeneous cytoplasm were selected with use of a stereomicroscope and washed three times in modified synthetic oviduct fluid medium (m-SOF) [15] supplemented with MEM nonessential amino acids (Invitrogen, Burlington, Ont., Canada), MEM essential amino acids (Invitrogen), 0.4 mM pyruvic acid, 1 mM glutamine (Sigma-Aldrich), and 50 mg/mL gentamicin (Sigma-Aldrich). The m-SOF medium used in this study was based upon the original formulation [15] with subsequent modifications [16]. 2.2. In vitro maturation The COCs were incubated (in groups of 10) in 50 mL droplets of maturation medium that consisted of m-SOF supplemented with 8 mg/mL polyvinylpyrrolidone (PVP-40; SigmaAldrich), 1 MEM nonessential amino acids, 1 MEM essential amino acids, 1.5 mM glucose (Sigma-Aldrich), 1 mM glutamine, and 50 mg/mL gentamicin. The droplets were covered with mineral oil (Sigma-Aldrich) and were pre-incubated under the maturation conditions for a minimum of 3 h (38.5 8C, 5% CO2 in air with 100% humidity) and then incubated for 24 h after oocytes were added. 2.3. In vitro fertilization In vitro fertilization took place in droplets (48 mL) composed of modified Tyrode lactate medium [17], supplemented with 0.6% BSA fatty acid free (Sigma-Aldrich), 0.2 mM pyruvic acid, 2 mg/mL heparin (Sigma-Aldrich), and 50 mg/mL gentamicin containing five oocytes. The COCs were previously washed twice for 5 min in Hepes buffered Tyrode’s medium (TLH). Following the transfer of oocytes, 2 mL of PHE (2 mM penicillamine, 1 mM hypotaurine, 250 mM epinephrine; Sigma-Aldrich) was added to each droplet. All experiments were carried out using frozen semen from the same bull (Centre d’Inse´ mination Artificielle du Que´ bec; CIAQ, St-Hyacinthe, PQ, Canada). Spermatozoa were thawed in a 35 8C water-bath for 1 min and were then washed in a discontinous Percoll gradient prepared by adding 2 mL of 90% Percoll under 2 mL of 45% Percoll in a 15-mL centrifuge tube (Falcon). The semen samples were added on top of the Percoll gradient and centrifuged at 700 g for 30 min at 26 8C. The pellet was removed and resuspended in 1 mL of modified Tyrode’s medium (TALP) and centrifuged at 250 g for 5 min at 26 8C. After removal of the supernatant, spermatozoa were resuspended in IVF medium, counted in a hemocytometer chamber and 2 mL of sperm suspension (final concentration ¼ 1 106 cells/mL) were added into each droplet. Incubation was carried out (for 15–18 h) at 38.5 8C in 5% CO2 in air with saturated humidity. 2.4. In vitro culture In all experiments, embryo culture took place in m-SOF [16,18] under mineral oil in a humidified atmosphere of 5% CO2, 7% O2, 88% N2 at 38.5 8C. From 15 to 18 h after
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insemination, COCs were denuded of surrounding cumulus cells by repeated pipetting in phosphate buffered saline (PBS) and subsequently washed three times in PBS before being transferred to the culture droplets (50 mL) in groups of 20–30 embryos. Cleavage was assessed after 72 h of culture and the number of embryos developing to blastocyst stage was assessed on Day 8. This study used a two-culture system as described previously [1,19]. The first system (SOFC1) medium contained 0.8% BSA-V, 1 MEM nonessential amino acids, 1 mM glutamine, and 10 mM EDTA (Sigma-Aldrich) for the first 72 h. Then, the medium was replaced by the second system (SOFC2) containing 0.8% BSA-V, 1 MEM nonessential amino acids, and 1 MEM essential amino acids and 1 mM glutamine for the remaining 96 h of culture. To prevent toxic accumulation of ammonia as a result of amino acid degradation, SOFC2 medium was replaced after 72 h of culture. 2.5. Follicular fluid source and collection The protocol described by Blondin et al. [20] was used without any change. In brief, six cyclic Holstein heifers were synchronized by inserting a progesteronereleasing device (CIDR; donated by Bioniche Animal Health Ltd., Belleville, Ont., Canada) for 9 days and injected with a prostaglandin F2 alpha analogue (Estrumate, 2 mL i.m.; Shering Canada, Pointe-Claire, PQ, Canada) on removal of the CIDR. Superstimulation was initiated between Days 6 and 10 of the estrous cycle, and the dominant follicle was aspirated 2 days before administration of hormones. The superstimulation regimen consisted of six constant doses of FSH (Folltropin, 300 mg i.m. total; donated by Bioniche Animal Health Ltd.) administered in constant doses every 12 h. In half the heifers, 25 mg of LH (Lutropin, Bioniche Animal Health Ltd) was administered i.v. 6 h before follicular aspiration. Follicular diameters were measured with a 5-MHz, transvaginal, Hitachi EUB-405 Plus Veterinary Ultrasound Transducer (Products Group International, Inc., Lyons, CO). For each heifer, bFF of follicles between 5 and 15 mm were aspirated and pooled 48 h after the last FSH injection (coasting period) using the ultrasound-guided transvaginal approach in vivo. The same follicular fluid was used for all experiments. The other follicular fluid was obtained from a pool of small follicles (2–5 mm) harvested from the ovaries of untreated cows. All samples were centrifuged at 14,000 g for 60 min at 4 8C, heated 30 min at 56 8C to destroy the complement and frozen at 80 8C until used. 2.6. Experimental design 2.6.1. Experiment 1 The objective of this experiment was to investigate the effect of different sources and concentrations of protein on bovine oocyte maturation and subsequent embryo development. The COCs were cultured in m-SOF in the presence of different concentrations (1, 5, and 10%) of bFF from competent follicles (>8 mm), or bFF from small follicles (2–5 mm), or BSA-V. The COCs were then fertilized and putative zygotes were cultured as described
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above. At the end of the 7th day of cultured (8 d.p.i), embryos were examined to evaluate development to the blastocyst stage. 2.6.2. Experiment 2 The objective of this experiment was to determine the effect of hormones in presence of bFF from competent (>8 mm) or small (2–5 mm) follicles on bovine oocyte maturation and subsequent embryo development. In this experiment, 0.5 mg/mL recombinant folliclestimulating hormone (r-hFSH; Serono Canada Inc., Oakville, Ont., Canada) and/or 1 mg/ mL estradiol (E2; Sigma-Aldrich) were added to maturation medium with or without 5% bFF (the concentration of bFF shown to promote a high rate of oocyte development in Experiment 1). The COCs were then fertilized and putative zygotes were cultured as described above. At the end of the 7th day of culture (8 d.p.i), embryos were examined to evaluate development to the blastocyst stage. 2.6.3. Experiment 3 The objective of this experiment was to determine the effect of r-hFSH and E2 in maturation medium containing bFF from competent (>8 mm) or small (2–5 mm) follicles on cumulus expansion, development rate to the blastocyst stage and quality of blastocysts obtained following in vitro fertilization. Only the concentration of bFF that had been shown to promote high rate of oocyte development in Experiment 1 was used. Thus, COCS were cultured in maturation medium containing 5% bFF, with or without 0.5 mg/mL r-hFSH and 1 mg/mL E2. At the end of the maturation period, cumulus expansion was evaluated under a stereomicroscope as described by Ali et al. [1]. The COCs were then fertilized and putative zygotes were cultured as described above. At the end of the 7th day of cultured (8 d.p.i), embryos were examined to evaluate blastocyst stage and quality. To evaluate quality of blastocysts, differential staining of inner cell mass (ICM) and trophectoderm (TE) was done and nuclei of each type were counted. Nuclei of ICM and TE were differentially labeled with propidium iodide (PI) and Hoechst (Sigma-Aldrich), respectively. The protocol described by Van Soom et al. [21] was used with two changes. Instead of TCM-199 Hepes, the TLH wash medium without BSA and gentamicin was used. For the coloration of the ICM by the Hoescht, blastocysts were fixed in 100 mL of formalin (SigmaAldrich) for 15 min, then rinsed in 200 mL 0.5% Triton X-100 (Sigma-Aldrich) before being mounted over a drop of Mowiol gelatin containing Hoescht 33354 (5 mg/mL) as described by Campagna et al. [22]. 2.7. Statistical analysis All experiments were replicated three times. For Experiments 1 and 3, statistical analysis was carried out by ANOVA with Fisher’s protected least significant difference (LSD) test using the STATVIEW program (SAS Institute, Inc., Cary, NC, USA). All percentage data were subjected to arcsine transformation before statistical analysis [23]. For Experiment 2, statistical analysis was carried out by 2-way ANOVA for each of the three follicular fluid conditions tested using the STATVIEW program without data transformation. Data from three replicates were expressed as mean S:E:M:. A probability of P < 0:05 was considered significant.
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3. Results 3.1. Experiment 1 The effect of supplementation of the maturation medium with bFF depended on the bFF concentration used (Fig. 1). Development to the blastocyst stage of oocytes matured in the presence of 5% bFF from competent follicles (>8 mm) was significantly greater than that of control oocytes or those matured in bFF derived from small follicles (2–5 mm). In contrast, the rate of development to the blastocyst stage was significantly reduced in the presence of all concentrations of bFF derived from small follicles and of 10% bFF from competent follicles. Also, the presence of BSA-V at all concentrations significantly reduced the developmental rate to the blastocyst stage. 3.2. Experiment 2 The effect of supplementation of the maturation medium with hormones in presence of 5% bFF on development rate depended on the source of bFF. In the absence of follicular fluid, addition of hormones had no significant effect (control). In presence of bFF from small follicles (2–5 mm), overall production of blastocysts was lower, but E2 or r-hFSH alone significantly increased the developmental rate to the blastocyst stage. In the presence of bFF from competent follicles (>8 mm), a combination of E2 and r-hFSH significantly 50 a
45 bFF or BSA (%)
ab
40
0
Blastocysts (%)
35
b
1 5
30
10 25 c
20
c
c
c c
c
c
15 10 5 0 Control
bFF (2 to 5 mm)
bFF (>8 mm)
BSA
Treatments
Fig. 1. Effect of various concentrations of bFF and BSA added to the IVM medium on the developmental capacity of fertilized oocytes. Pooled data from three replicates of 20 oocytes each (mean S:E:M:). Values with different superscripts (a, b) are different (P < 0:05).
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Fig. 2. Effects of 1 mg/mL E2 and/or 0.5 mg/mL r-hFSH added to 5% bFF from small follicles (2–5 mm) or from competent follicles (>8 mm) during IVM of bovine oocytes on embryo development. Pooled data from three replicates of 20 oocytes each (mean S:E:M:). For the same follicular fluid condition (control, 2–5 mm or >8 mm), values with different superscripts (a, b) are different (P < 0:05).
increased development to the blastocyst stage, indicating a significant synergism between these two hormones (Fig. 2). 3.3. Experiment 3 The effect of r-hFSH and E2 added to bFF on cumulus expansion, development rate to the blastocyst stage and quality of blastocysts are shown in Table 1. Addition of 0.5 mg/mL r-hFSH and 1 mg/mL E2 to the maturation medium in the presence of 5% bFF from competent follicles (>8 mm) yielded a high development rate but no cumulus expansion was noted, while in the presence of bFF derived from small follicles (2–5 mm), cumulus expansion was stimulated but development rate to the blastocyst stage was low. Addition of r-hFSH and E2 to media containing bFF from competent follicles significantly increased blastocyst expansion and hatching compared to the addition of hormones to media supplemented with bFF derived from small follicles. Moreover, addition of r-hFSH and E2 to media supplemented with bFF from competent follicles significantly increased the cell numbers of blastocysts, the number of ICM cells and the percentage of ICM/total cell compared to the addition of r-hFSH and E2 to media supplemented with bFF derived from small follicles. No differences between treatments were observed regarding general appearance of the embryos.
4. Discussion To our knowledge, this is the first report of such high developmental rates after in vitro maturation, especially after supplementation of bFF. In the present study, modified SOF
Treatment
Control r-hFSH þ E2 BFF (>8 mm) BFF (>8 mm) þ r-hFSHþE2 BFF (2–5 mm) BFF (2–5 mm) þ r-hFSHþE2
Oocytes (n)
Cumulus expansion
Blastocysts
58 53 58 55
þ
12.80 13.20 9.90 5.00
56 53
þ þþþ
With cavity
8.0a 2.0a 3.2a,b 2.1c
8.40 2.4a,b 6.02 0.8c
Expanded 9.60 10.34 13.30 18.25
0.8b 1.9b 0.8a,b 2.8a
5.60 1.0c 5.70 1.7c
Blastocyst cell number
Hatched 7.60 10.34 13.80 33.75
2.9b 2.4b 2.0b 3.3a
0 14.28 2.0b
129.99 137.99 133.93 151.02
7.2b 4.5b 6.0b 2.8a
117.88 8.5b 120.30 6.5b
ICM cell number 32.28 34.33 44.87 51.30
2.7b 1.7b 2.3a,b 3.5a
25.88 0.3c 25.50 3.7c
Trophectoderm cell number 97.71 103.66 89.06 99.72
4.0a 1.5a 1.5a 2.5a
92.00 5.7a 94.80 4.7a
ICM/total (%) 24.83 24.87 33.50 33.39
2.3b 1.4b 1.8a 0.0a
21.95 3.9c 21.19 6.0c
Pooled data from three replicates of 20 oocytes each (mean S:E:M:). Values with different superscripts (a–c) in same column are different (P < 0:05).
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Table 1 Effects of 0.5 mg/mL r-hFSH and 1 mg/mL E2 added to 5% bFF from competent follicles (>8 mm) or from small follicles (2–5 mm) during IVM of bovine oocytes on cumulus expansion and embryonic quality after in vitro fertilization
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was used successfully to mature and culture bovine oocytes and embryos, resulting in development (rate and quality) that approached what is observed in vivo. Furthermore, to our knowledge, this is the first demonstration that combined treatment with FSH and E2 has a greater benefit than treatment with FSH alone. It is believed that induction of a preovulatory-type follicular environment is necessary to trigger COC to complete their cytoplasmic maturation [20]. The protein concentration in follicular fluid is of the same order of magnitude as in serum; therefore, oocytes tolerate a microenvironment high in protein. To verify the possible effect of a novel component from follicles, the bFF derived from treated and coasted follicles (>8 mm), a pool of small follicles (2–5 mm) or BSA-V were used in the IVM medium as a source of protein supplement. The effect of supplementation of the maturation medium with bFF depended on the bFF concentration used. Supplementation of the IVM medium with competent bFF (5%) was favorable to the acquisition of developmental competence immature oocytes, since a higher (P < 0:05) proportion of oocytes developed to the blastocyst stage after in vitro fertilization, compared with bFF derived from small follicles or BSA-V. This is a strong indication of the importance of the follicle influencing developmental competence, and that protein supplementation during in vitro maturation can have profound effects on both the rate and efficiency of development. Moreover, in this study, the use of BSA-Vat all concentrations as the only protein source during IVM of bovine oocytes decreased the developmental capacity of the oocytes, as reflected by the blastocyst yield after IVF, was in agreement with our recent study [18]. The process of cumulus expansion is an apparently important step during IVM; optimal expansion of the cumulus mass appears to be essential for both ovulation and subsequent fertilization up to blastocyst stage [24–28]. When oocytes were cultured in SOF with PVP in the present study, no cumulus expansion was observed. Surprisingly, cumulus expansion was never detected in the present study when COCs were matured in maturation medium supplemented with competent bFF with r-hFSH and E2 compared to maximal expansion observed with maturation medium supplemented with bFF derived from small follicles. In this case, the absence of cumulus expansion did not adversely affect development of the resulting embryos to the blastocyst stage. These results confirmed our recent findings that the presence of cumulus cells during IVM of the bovine oocyte might be necessary to express the competence of oocyte, but cumulus expansion is not required to improve competence; at least there is no linear relationship between cumulus expansion and cytoplasmic maturation [1]. We recently demonstrated that the effect of FSH on nuclear maturation and cumulus expansion was dependent on molecules present in the IVM medium [1]. Another study in our laboratory has shown that the presence of physiological concentrations of E2 and rhFSH during IVM of bovine oocytes in defined medium, significantly improved oocyte quality, as indicated by the increase in the proportion of oocytes developing to the blastocyst stage after IVF [19]. It is interesting to note that in the present study, addition of r-hFSH and E2 to competent bFF during IVM of bovine oocytes, enhanced (P < 0:05) oocyte quality, as indicated by the increase in the proportion of oocytes developing to the blastocyst stage after IVF. Follicular fluid originating from follicles containing competent oocytes produced and released signals that increased the developmental competence of oocytes, resulting in the superior blastocyst yield in this study.
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Another approach to study the effect of bFF during in vitro maturation is to examine embryonic quality after in vitro fertilization. It is unknown at this stage whether the presence of bFF during the maturation period as a source of protein actually increased viability of the resulting embryos or simply increased the rate of cell division. Although, it is recognized that embryo morphology may not be a good predictor of embryo viability [29,30], the examination of inner cells mass:trophectoderm ratios is believed to be a better indication of embryo quality. Inclusion of competent bFF with r-hFSH and E2 during maturation increased total cell numbers, ICM, and the ICM:trophectoderm ratio compared to oocytes matured in the presence of bFF from small follicles. A substantial proportion (approximately 32%) of an in vivo produced hatched blastocyst is occupied by the ICM [31], which is in agreement with our present study, emphasizing the importance of the in vitro maturation environment on subsequent development. Hatching was also significantly improved when COCs were cultured with competent bFF with r-hFSH and E2, compared to those matured in the presence of bFF derived from small follicles. These results suggested that bFF originating from follicles containing competent oocytes had greater developmental potential than bFF derived from small follicles, and that the origin of bFF, which is added to the maturation media as a source of protein, might be an important factor for improving in vitro development of bovine oocytes. In conclusion, the presence of bFF from competent follicles as a source of protein and the SOF system supplemented with E2 and r-hFSH increased blastocyst numbers and improved embryonic quality to levels approaching those characteristic of in vivo matured oocytes.
Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).
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Origin of bovine follicular fluid and its effect during in vitro maturation on the developmental competence of bovine oocytes Atef Alia, Karine Coenena, Daniel Bousquetb, Marc-Andre´ Sirarda,* a
Department of Animal Science, Centre de Recherche en Biologie de la Reproduction (CRBR), Laval University, Ste-Foy, Que., Canada G1K 7P4 b L’Alliance Boviteq Inc., St-Hyacinthe, Que., Canada J2S 7A9 Received 7 July 2003; accepted 3 March 2004
Abstract Protein supplementation during in vitro maturation can profoundly affect both the rate and overall efficiency of the maturation procedure [Biol. Reprod. 66 (2002) 901]. The present study was conducted to assess the ability of different concentrations (1, 5, and 10%) of bovine follicular fluid (bFF) to support in vitro maturation of oocytes and subsequent developmental capacity. The bFF was derived either from competent follicles (>8 mm) obtained by transvaginal recovery following superovulation or from a pool of small follicles (2–5 mm) from abbatoir-derived ovaries. Bovine oocytes were cultured for 24 h in synthetic oviduct fluid medium (m-SOF) supplemented with polyvinylpyrrolidone. Following fertilization and embryo culture, more oocytes (P < 0:05) reached the blastocyst stage when oocytes were cultured with 5% bFF from competent follicles (41 3:7%) compared with bFF derived from small follicles (16 2:9%). Estradiol and recombinant human follicle stimulating hormone added to the competent bFF during maturation acted in synergy to increase blastocyst production rate (P < 0:05); this blastocyst production rate (57 1:2%) was higher than those obtained with the addition of these two hormones to bFF derived from small follicles (26 2:9%). The quality of blastocysts obtained was reflected by inner cell mass (51:30 3:5 and 25:50 3:7) and trophectoderm cell numbers (99:72 2:5 and 94:80 4:7) for bFF from competent and small follicles, respectively. In conclusion, follicular fluid originating from competent follicles increased the developmental competence of abbatoir-derived oocytes. # 2004 Elsevier Inc. All rights reserved. Keywords: Bovine oocyte; Follicle; Early development; In vitro fertilization; Embryo
* Corresponding author. Fax: þ1 418 656 3766. E-mail address: [email protected] (M.-A. Sirard).
0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.03.011
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1. Introduction When comparing oocytes matured in vivo versus in vitro, no apparent differences are evident in the level of nuclear maturation, in the rates of in vitro fertilization or cleavage, but rather in the in vitro developmental competence of the oocytes [2,3]. Better cytoplasmic maturation of oocytes matured in vivo could explain the higher developmental competence observed [2]. Bovine oocytes resuming meiosis in vivo originate from dominant follicles about 15 mm that have been grown from 4 to 15 mm in approximately 5 days [4]. In contrast, oocytes for in vitro maturation (IVM) are usually retrieved from 2 to 6 mm follicles that are between 4 and 10 days from potential ovulation and the IVM period lasts only 24 h [5]. Thus, it is possible that oocytes matured in vitro have not acquired maximal developmental competence due to incomplete oocyte capacitation, a process during which RNA, proteins, and other molecules are synthesized [6]. Intrinsic factors such as the size of the follicle of origin could influence the percentage of bovine embryos produced in vitro [5,7,8]. Larger follicles contain oocytes more likely to develop into blastocysts than oocytes from smaller follicles. How the ovaries were handled before the culture is also an important factor that can affect the expression of developmental competence. Blondin et al. [9] found that bovine oocytes derived from ovaries maintained at 35 8C for 4 h after slaughter yielded higher frequencies of blastocyst development than those collected 2, 6 or 7 h post-slaughter, thus indicating that some developmental competence may be acquired shortly prior to in vitro maturation. Our laboratory has also demonstrated that the ‘‘coasting’’ period between hormonal stimulation and ovary collection [10] as well as the time interval from ovary collection to oocyte aspiration [9] significantly affected the developmental potential of COC. Addition of selected bovine follicular fluid (bFF) to the maturation medium influenced the developmental potential of oocytes obtained from unselected ovaries [11–14]. The use of bFF as a supplement to the maturation medium generally resulted in improved cytoplasmic maturation (to an extent similar to that achieved with fetal calf serum). The effect of bFF is not always affected by follicle size but may be affected by the quality offollicle from which it is obtained [12,14]. Exposure of oocytes to bFF collected at different times after LH surge profoundly affects rates of blastocyst formation [13]. In fact, the beneficial effect on development was present in dominant follicles, but not in all growing or regressing dominant follicles [11], suggesting subtle differences between growth and differentiation signals. It appears that follicular fluid influences oocyte competence, developmental capacity, and embryonic quality. To test this hypothesis, COCs were incubated in completely defined maturation medium supplemented with various concentrations of bFF derived from competent follicles (>8 mm) or small follicles (2–5 mm), or bovine serum albumin fraction V (BSA-V; as a source of nonspecific protein). 2. Materials and methods 2.1. Oocyte recovery and selection Bovine ovaries at various stages of their estrous cycle were obtained from a abbatoir and transported to the laboratory in a 0.9% NaCl aqueous solution containing 100,000 IU/L
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penicillin, 100 mg/L streptomycin, and 250 mg/L amphotericin B (Sigma-Aldrich, Oakville, Ont., Canada). Cumulus-oocyte complexes (COCs) were aspirated from 2 to 6 mm follicles with an 18-g needle attached to a 10-mL syringe. Only oocytes with 4 layers of compact cumulus cells and homogeneous cytoplasm were selected with use of a stereomicroscope and washed three times in modified synthetic oviduct fluid medium (m-SOF) [15] supplemented with MEM nonessential amino acids (Invitrogen, Burlington, Ont., Canada), MEM essential amino acids (Invitrogen), 0.4 mM pyruvic acid, 1 mM glutamine (Sigma-Aldrich), and 50 mg/mL gentamicin (Sigma-Aldrich). The m-SOF medium used in this study was based upon the original formulation [15] with subsequent modifications [16]. 2.2. In vitro maturation The COCs were incubated (in groups of 10) in 50 mL droplets of maturation medium that consisted of m-SOF supplemented with 8 mg/mL polyvinylpyrrolidone (PVP-40; SigmaAldrich), 1 MEM nonessential amino acids, 1 MEM essential amino acids, 1.5 mM glucose (Sigma-Aldrich), 1 mM glutamine, and 50 mg/mL gentamicin. The droplets were covered with mineral oil (Sigma-Aldrich) and were pre-incubated under the maturation conditions for a minimum of 3 h (38.5 8C, 5% CO2 in air with 100% humidity) and then incubated for 24 h after oocytes were added. 2.3. In vitro fertilization In vitro fertilization took place in droplets (48 mL) composed of modified Tyrode lactate medium [17], supplemented with 0.6% BSA fatty acid free (Sigma-Aldrich), 0.2 mM pyruvic acid, 2 mg/mL heparin (Sigma-Aldrich), and 50 mg/mL gentamicin containing five oocytes. The COCs were previously washed twice for 5 min in Hepes buffered Tyrode’s medium (TLH). Following the transfer of oocytes, 2 mL of PHE (2 mM penicillamine, 1 mM hypotaurine, 250 mM epinephrine; Sigma-Aldrich) was added to each droplet. All experiments were carried out using frozen semen from the same bull (Centre d’Inse´ mination Artificielle du Que´ bec; CIAQ, St-Hyacinthe, PQ, Canada). Spermatozoa were thawed in a 35 8C water-bath for 1 min and were then washed in a discontinous Percoll gradient prepared by adding 2 mL of 90% Percoll under 2 mL of 45% Percoll in a 15-mL centrifuge tube (Falcon). The semen samples were added on top of the Percoll gradient and centrifuged at 700 g for 30 min at 26 8C. The pellet was removed and resuspended in 1 mL of modified Tyrode’s medium (TALP) and centrifuged at 250 g for 5 min at 26 8C. After removal of the supernatant, spermatozoa were resuspended in IVF medium, counted in a hemocytometer chamber and 2 mL of sperm suspension (final concentration ¼ 1 106 cells/mL) were added into each droplet. Incubation was carried out (for 15–18 h) at 38.5 8C in 5% CO2 in air with saturated humidity. 2.4. In vitro culture In all experiments, embryo culture took place in m-SOF [16,18] under mineral oil in a humidified atmosphere of 5% CO2, 7% O2, 88% N2 at 38.5 8C. From 15 to 18 h after
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insemination, COCs were denuded of surrounding cumulus cells by repeated pipetting in phosphate buffered saline (PBS) and subsequently washed three times in PBS before being transferred to the culture droplets (50 mL) in groups of 20–30 embryos. Cleavage was assessed after 72 h of culture and the number of embryos developing to blastocyst stage was assessed on Day 8. This study used a two-culture system as described previously [1,19]. The first system (SOFC1) medium contained 0.8% BSA-V, 1 MEM nonessential amino acids, 1 mM glutamine, and 10 mM EDTA (Sigma-Aldrich) for the first 72 h. Then, the medium was replaced by the second system (SOFC2) containing 0.8% BSA-V, 1 MEM nonessential amino acids, and 1 MEM essential amino acids and 1 mM glutamine for the remaining 96 h of culture. To prevent toxic accumulation of ammonia as a result of amino acid degradation, SOFC2 medium was replaced after 72 h of culture. 2.5. Follicular fluid source and collection The protocol described by Blondin et al. [20] was used without any change. In brief, six cyclic Holstein heifers were synchronized by inserting a progesteronereleasing device (CIDR; donated by Bioniche Animal Health Ltd., Belleville, Ont., Canada) for 9 days and injected with a prostaglandin F2 alpha analogue (Estrumate, 2 mL i.m.; Shering Canada, Pointe-Claire, PQ, Canada) on removal of the CIDR. Superstimulation was initiated between Days 6 and 10 of the estrous cycle, and the dominant follicle was aspirated 2 days before administration of hormones. The superstimulation regimen consisted of six constant doses of FSH (Folltropin, 300 mg i.m. total; donated by Bioniche Animal Health Ltd.) administered in constant doses every 12 h. In half the heifers, 25 mg of LH (Lutropin, Bioniche Animal Health Ltd) was administered i.v. 6 h before follicular aspiration. Follicular diameters were measured with a 5-MHz, transvaginal, Hitachi EUB-405 Plus Veterinary Ultrasound Transducer (Products Group International, Inc., Lyons, CO). For each heifer, bFF of follicles between 5 and 15 mm were aspirated and pooled 48 h after the last FSH injection (coasting period) using the ultrasound-guided transvaginal approach in vivo. The same follicular fluid was used for all experiments. The other follicular fluid was obtained from a pool of small follicles (2–5 mm) harvested from the ovaries of untreated cows. All samples were centrifuged at 14,000 g for 60 min at 4 8C, heated 30 min at 56 8C to destroy the complement and frozen at 80 8C until used. 2.6. Experimental design 2.6.1. Experiment 1 The objective of this experiment was to investigate the effect of different sources and concentrations of protein on bovine oocyte maturation and subsequent embryo development. The COCs were cultured in m-SOF in the presence of different concentrations (1, 5, and 10%) of bFF from competent follicles (>8 mm), or bFF from small follicles (2–5 mm), or BSA-V. The COCs were then fertilized and putative zygotes were cultured as described
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above. At the end of the 7th day of cultured (8 d.p.i), embryos were examined to evaluate development to the blastocyst stage. 2.6.2. Experiment 2 The objective of this experiment was to determine the effect of hormones in presence of bFF from competent (>8 mm) or small (2–5 mm) follicles on bovine oocyte maturation and subsequent embryo development. In this experiment, 0.5 mg/mL recombinant folliclestimulating hormone (r-hFSH; Serono Canada Inc., Oakville, Ont., Canada) and/or 1 mg/ mL estradiol (E2; Sigma-Aldrich) were added to maturation medium with or without 5% bFF (the concentration of bFF shown to promote a high rate of oocyte development in Experiment 1). The COCs were then fertilized and putative zygotes were cultured as described above. At the end of the 7th day of culture (8 d.p.i), embryos were examined to evaluate development to the blastocyst stage. 2.6.3. Experiment 3 The objective of this experiment was to determine the effect of r-hFSH and E2 in maturation medium containing bFF from competent (>8 mm) or small (2–5 mm) follicles on cumulus expansion, development rate to the blastocyst stage and quality of blastocysts obtained following in vitro fertilization. Only the concentration of bFF that had been shown to promote high rate of oocyte development in Experiment 1 was used. Thus, COCS were cultured in maturation medium containing 5% bFF, with or without 0.5 mg/mL r-hFSH and 1 mg/mL E2. At the end of the maturation period, cumulus expansion was evaluated under a stereomicroscope as described by Ali et al. [1]. The COCs were then fertilized and putative zygotes were cultured as described above. At the end of the 7th day of cultured (8 d.p.i), embryos were examined to evaluate blastocyst stage and quality. To evaluate quality of blastocysts, differential staining of inner cell mass (ICM) and trophectoderm (TE) was done and nuclei of each type were counted. Nuclei of ICM and TE were differentially labeled with propidium iodide (PI) and Hoechst (Sigma-Aldrich), respectively. The protocol described by Van Soom et al. [21] was used with two changes. Instead of TCM-199 Hepes, the TLH wash medium without BSA and gentamicin was used. For the coloration of the ICM by the Hoescht, blastocysts were fixed in 100 mL of formalin (SigmaAldrich) for 15 min, then rinsed in 200 mL 0.5% Triton X-100 (Sigma-Aldrich) before being mounted over a drop of Mowiol gelatin containing Hoescht 33354 (5 mg/mL) as described by Campagna et al. [22]. 2.7. Statistical analysis All experiments were replicated three times. For Experiments 1 and 3, statistical analysis was carried out by ANOVA with Fisher’s protected least significant difference (LSD) test using the STATVIEW program (SAS Institute, Inc., Cary, NC, USA). All percentage data were subjected to arcsine transformation before statistical analysis [23]. For Experiment 2, statistical analysis was carried out by 2-way ANOVA for each of the three follicular fluid conditions tested using the STATVIEW program without data transformation. Data from three replicates were expressed as mean S:E:M:. A probability of P < 0:05 was considered significant.
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3. Results 3.1. Experiment 1 The effect of supplementation of the maturation medium with bFF depended on the bFF concentration used (Fig. 1). Development to the blastocyst stage of oocytes matured in the presence of 5% bFF from competent follicles (>8 mm) was significantly greater than that of control oocytes or those matured in bFF derived from small follicles (2–5 mm). In contrast, the rate of development to the blastocyst stage was significantly reduced in the presence of all concentrations of bFF derived from small follicles and of 10% bFF from competent follicles. Also, the presence of BSA-V at all concentrations significantly reduced the developmental rate to the blastocyst stage. 3.2. Experiment 2 The effect of supplementation of the maturation medium with hormones in presence of 5% bFF on development rate depended on the source of bFF. In the absence of follicular fluid, addition of hormones had no significant effect (control). In presence of bFF from small follicles (2–5 mm), overall production of blastocysts was lower, but E2 or r-hFSH alone significantly increased the developmental rate to the blastocyst stage. In the presence of bFF from competent follicles (>8 mm), a combination of E2 and r-hFSH significantly 50 a
45 bFF or BSA (%)
ab
40
0
Blastocysts (%)
35
b
1 5
30
10 25 c
20
c
c
c c
c
c
15 10 5 0 Control
bFF (2 to 5 mm)
bFF (>8 mm)
BSA
Treatments
Fig. 1. Effect of various concentrations of bFF and BSA added to the IVM medium on the developmental capacity of fertilized oocytes. Pooled data from three replicates of 20 oocytes each (mean S:E:M:). Values with different superscripts (a, b) are different (P < 0:05).
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Fig. 2. Effects of 1 mg/mL E2 and/or 0.5 mg/mL r-hFSH added to 5% bFF from small follicles (2–5 mm) or from competent follicles (>8 mm) during IVM of bovine oocytes on embryo development. Pooled data from three replicates of 20 oocytes each (mean S:E:M:). For the same follicular fluid condition (control, 2–5 mm or >8 mm), values with different superscripts (a, b) are different (P < 0:05).
increased development to the blastocyst stage, indicating a significant synergism between these two hormones (Fig. 2). 3.3. Experiment 3 The effect of r-hFSH and E2 added to bFF on cumulus expansion, development rate to the blastocyst stage and quality of blastocysts are shown in Table 1. Addition of 0.5 mg/mL r-hFSH and 1 mg/mL E2 to the maturation medium in the presence of 5% bFF from competent follicles (>8 mm) yielded a high development rate but no cumulus expansion was noted, while in the presence of bFF derived from small follicles (2–5 mm), cumulus expansion was stimulated but development rate to the blastocyst stage was low. Addition of r-hFSH and E2 to media containing bFF from competent follicles significantly increased blastocyst expansion and hatching compared to the addition of hormones to media supplemented with bFF derived from small follicles. Moreover, addition of r-hFSH and E2 to media supplemented with bFF from competent follicles significantly increased the cell numbers of blastocysts, the number of ICM cells and the percentage of ICM/total cell compared to the addition of r-hFSH and E2 to media supplemented with bFF derived from small follicles. No differences between treatments were observed regarding general appearance of the embryos.
4. Discussion To our knowledge, this is the first report of such high developmental rates after in vitro maturation, especially after supplementation of bFF. In the present study, modified SOF
Treatment
Control r-hFSH þ E2 BFF (>8 mm) BFF (>8 mm) þ r-hFSHþE2 BFF (2–5 mm) BFF (2–5 mm) þ r-hFSHþE2
Oocytes (n)
Cumulus expansion
Blastocysts
58 53 58 55
þ
12.80 13.20 9.90 5.00
56 53
þ þþþ
With cavity
8.0a 2.0a 3.2a,b 2.1c
8.40 2.4a,b 6.02 0.8c
Expanded 9.60 10.34 13.30 18.25
0.8b 1.9b 0.8a,b 2.8a
5.60 1.0c 5.70 1.7c
Blastocyst cell number
Hatched 7.60 10.34 13.80 33.75
2.9b 2.4b 2.0b 3.3a
0 14.28 2.0b
129.99 137.99 133.93 151.02
7.2b 4.5b 6.0b 2.8a
117.88 8.5b 120.30 6.5b
ICM cell number 32.28 34.33 44.87 51.30
2.7b 1.7b 2.3a,b 3.5a
25.88 0.3c 25.50 3.7c
Trophectoderm cell number 97.71 103.66 89.06 99.72
4.0a 1.5a 1.5a 2.5a
92.00 5.7a 94.80 4.7a
ICM/total (%) 24.83 24.87 33.50 33.39
2.3b 1.4b 1.8a 0.0a
21.95 3.9c 21.19 6.0c
Pooled data from three replicates of 20 oocytes each (mean S:E:M:). Values with different superscripts (a–c) in same column are different (P < 0:05).
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Table 1 Effects of 0.5 mg/mL r-hFSH and 1 mg/mL E2 added to 5% bFF from competent follicles (>8 mm) or from small follicles (2–5 mm) during IVM of bovine oocytes on cumulus expansion and embryonic quality after in vitro fertilization
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was used successfully to mature and culture bovine oocytes and embryos, resulting in development (rate and quality) that approached what is observed in vivo. Furthermore, to our knowledge, this is the first demonstration that combined treatment with FSH and E2 has a greater benefit than treatment with FSH alone. It is believed that induction of a preovulatory-type follicular environment is necessary to trigger COC to complete their cytoplasmic maturation [20]. The protein concentration in follicular fluid is of the same order of magnitude as in serum; therefore, oocytes tolerate a microenvironment high in protein. To verify the possible effect of a novel component from follicles, the bFF derived from treated and coasted follicles (>8 mm), a pool of small follicles (2–5 mm) or BSA-V were used in the IVM medium as a source of protein supplement. The effect of supplementation of the maturation medium with bFF depended on the bFF concentration used. Supplementation of the IVM medium with competent bFF (5%) was favorable to the acquisition of developmental competence immature oocytes, since a higher (P < 0:05) proportion of oocytes developed to the blastocyst stage after in vitro fertilization, compared with bFF derived from small follicles or BSA-V. This is a strong indication of the importance of the follicle influencing developmental competence, and that protein supplementation during in vitro maturation can have profound effects on both the rate and efficiency of development. Moreover, in this study, the use of BSA-Vat all concentrations as the only protein source during IVM of bovine oocytes decreased the developmental capacity of the oocytes, as reflected by the blastocyst yield after IVF, was in agreement with our recent study [18]. The process of cumulus expansion is an apparently important step during IVM; optimal expansion of the cumulus mass appears to be essential for both ovulation and subsequent fertilization up to blastocyst stage [24–28]. When oocytes were cultured in SOF with PVP in the present study, no cumulus expansion was observed. Surprisingly, cumulus expansion was never detected in the present study when COCs were matured in maturation medium supplemented with competent bFF with r-hFSH and E2 compared to maximal expansion observed with maturation medium supplemented with bFF derived from small follicles. In this case, the absence of cumulus expansion did not adversely affect development of the resulting embryos to the blastocyst stage. These results confirmed our recent findings that the presence of cumulus cells during IVM of the bovine oocyte might be necessary to express the competence of oocyte, but cumulus expansion is not required to improve competence; at least there is no linear relationship between cumulus expansion and cytoplasmic maturation [1]. We recently demonstrated that the effect of FSH on nuclear maturation and cumulus expansion was dependent on molecules present in the IVM medium [1]. Another study in our laboratory has shown that the presence of physiological concentrations of E2 and rhFSH during IVM of bovine oocytes in defined medium, significantly improved oocyte quality, as indicated by the increase in the proportion of oocytes developing to the blastocyst stage after IVF [19]. It is interesting to note that in the present study, addition of r-hFSH and E2 to competent bFF during IVM of bovine oocytes, enhanced (P < 0:05) oocyte quality, as indicated by the increase in the proportion of oocytes developing to the blastocyst stage after IVF. Follicular fluid originating from follicles containing competent oocytes produced and released signals that increased the developmental competence of oocytes, resulting in the superior blastocyst yield in this study.
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Another approach to study the effect of bFF during in vitro maturation is to examine embryonic quality after in vitro fertilization. It is unknown at this stage whether the presence of bFF during the maturation period as a source of protein actually increased viability of the resulting embryos or simply increased the rate of cell division. Although, it is recognized that embryo morphology may not be a good predictor of embryo viability [29,30], the examination of inner cells mass:trophectoderm ratios is believed to be a better indication of embryo quality. Inclusion of competent bFF with r-hFSH and E2 during maturation increased total cell numbers, ICM, and the ICM:trophectoderm ratio compared to oocytes matured in the presence of bFF from small follicles. A substantial proportion (approximately 32%) of an in vivo produced hatched blastocyst is occupied by the ICM [31], which is in agreement with our present study, emphasizing the importance of the in vitro maturation environment on subsequent development. Hatching was also significantly improved when COCs were cultured with competent bFF with r-hFSH and E2, compared to those matured in the presence of bFF derived from small follicles. These results suggested that bFF originating from follicles containing competent oocytes had greater developmental potential than bFF derived from small follicles, and that the origin of bFF, which is added to the maturation media as a source of protein, might be an important factor for improving in vitro development of bovine oocytes. In conclusion, the presence of bFF from competent follicles as a source of protein and the SOF system supplemented with E2 and r-hFSH increased blastocyst numbers and improved embryonic quality to levels approaching those characteristic of in vivo matured oocytes.
Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).
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