Modulatory Actions of Activin-A and Follistatin on the Developmental ...

BIOLOGY OF REPRODUCTION 58, 558-565 (1998)

Modulatory Actions of Activin-A and Follistatin on the Developmental Competence of In Vitro-Matured Bovine Oocytes' Celia C. Silva and Philip G. Knight2 School of Animal and Microbial Sciences, The University of Reading, Whiteknights, Reading RG6 6AJ, United Kingdom ABSTRACT

Reduced developmental capacity of in vitro-matured oocytes is believed to be due to deficient cytoplasmic maturation [4-7]. Thus, in addition to nuclear maturation, normal cytoplasmic maturation of oocytes must occur in order to produce a viable embryo. Several studies have suggested that specific follicular components can improve oocyte maturation [8-11]. Also, in vitro oocyte maturation is significantly improved by the presence of granulosa cumulus cells [12-15]. However, the mechanism by which follicular and granulosa cell factors influence the process of oocyte maturation remains to be clarified. Granulosa cells, and in particular cumulus cells, are a major site of activin and follistatin expression [16, 17]. Furthermore, activin receptor mRNA is expressed in oocytes and granulosa cells [18-20], suggesting that activin and its binding protein, follistatin, may exert autocrine/paracrine roles in the regulation of oocyte maturation. Several attempts have been made to assess the effect of activin and follistatin on oocyte maturation, but the results so far have been conflicting. In the majority of studies, observations have been restricted to effects on nuclear maturation. While some authors showed no effect of activin on nuclear maturation in rat [21, 22] and bovine [23] oocytes, others reported that activin increased the percentage of germinal vesicle breakdown in rat [19, 24] and primate [25] oocytes. In these studies, only nuclear maturation was assessed, and cytoplasmic maturation, as indicated by the ability of fertilized oocytes to develop to the blastocyst stage, was not monitored. Recently, Izadyar et al. [26] reported that addition of activin-A to bovine oocyte maturation media did not affect the proportion of blastocysts formed, but the dose of activin-A used (10 ng/ml) was very low compared with the endogenous levels (-3 ,ug/ml) recently shown to be present in bovine follicular fluid (bFF) [27]. The present study was performed to investigate the effects of more physiological levels of activin-A and follistatin on maturation of bovine cumulus-oocyte complexes (COCs). Also, oocytes stripped from their cumulus cells were used to evaluate any direct effects of these hormones on oocytes without the mediation of cumulus cells. In addition, the endogenous levels of activin-A and follistatin produced by groups of COCs during maturation in the absence of any treatment were measured and correlated with their subsequent developmental competence.

The presence of activin receptors on oocytes and granulosa cells suggests that activin and its binding protein, follistatin, may regulate oocyte maturation. The aim of the present study was to investigate whether activin-A and follistatin can influence the in vitro maturation of bovine oocytes as assessed by their competence to form blastocysts after in vitro fertilization. Bovine cumulus oocyte complexes (COCs) were cultured for 22-24 h at 38.5°C in tissue culture medium-199 supplemented with 10% estrous cow serum, eCG (2.5 IU/ml), and either no treatment (control), activin-A (0.1 or 0.5 tig/ml), follistatin (0.1, 1, or 10 tIg/ml), or activin-A (0.5 tIg/ml) in combination with follistatin (0.5 or 5 ,tg/ml). In separate experiments, the same treatments were also tested on cumulus-free oocytes, which had a much reduced developmental capacity when compared to COCs. Neither activin-A nor follistatin affected the postfertilization cleavage rate of either COCs (-60%) or cumulus-free oocytes (-40%). Activin increased (p < 0.05) the proportion of cleaved oocytes that reached the blastocyst stage when added to both COCs (38% increase) and cumulus-free oocytes (160% increase), with the magnitude of response much greater with the latter. Follistatin had a dose-dependent inhibitory effect on blastocyst yield from COCs (67% reduction, p < 0.05) and opposed the stimulatory effect of activin (p < 0.05). With cumulus-free oocytes, however, follistatin did not further decrease the low developmental potential of oocytes. A positive correlation (r = 0.57, p < 0.001) was found between endogenous levels of activin-A produced by COCs and their postcleavage development to the blastocyst stage. No such correlation was found between endogenous follistatin level and postcleavage development (r = 0.10, p = 0.46). Endogenous levels of activin-A and follistatin secreted by cumulus-free oocytes were undetectable. These in vitro observations support the hypothesis that activin-A and follistatin, both secretory products of cumulus cells, contribute to the regulation of oocyte maturation in vivo. INTRODUCTION In cattle, the preovulatory surge of LH triggers the maturation of the oocyte contained in the dominant follicle. During its maturation, the oocyte resumes meiosis and, in addition, its cytoplasm accumulates nutrients and organelles necessary to support the early stages of embryonic development. Under suitable conditions, bovine oocytes can acquire full developmental capacity after in vitro maturation as demonstrated by the production of live calves [1, 2]. However, despite all the improvements in culture conditions, there is still a clear difference in developmental competence between oocytes obtained from in vivo maturation and oocytes matured in culture [3, 4].

MATERIALS AND METHODS Culture Media The washing medium (WM) consisted of tissue culture medium (TCM)-199 (with Earle's salts, sodium bicarbonate and 25 mM HEPES buffer; Sigma, UK, Poole, Dorset, UK) supplemented with 10% heat-inactivated calf serum (Sigma), penicillin (50 IU/ml), and streptomycin (50 g/ml). The maturation medium (MM) was similar to that used by Funston and Seidel [28] with minor modifications [29]. It consisted of TCM-199 (with Earle's salts and sodium bi-

Accepted October 2, 1997. Received June 26, 1997. 'This research was supported by PRAXIS XXI/BD/3337/94-JNICT and BBSRC (grant S05760). 'Correspondence: P.G. Knight, School of Animal and Microbial Sciences, The University of Reading, PO Box 228, Reading RG6 6AJ, UK. FAX: 118 931 0180.

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ACTIVIN, FOLLISTATIN, AND BOVINE OOCYTE MATURATION carbonate; Sigma) supplemented with 10% estrous cow serum, L-glutamine (0.4 mM), pyruvate (0.2 mM), penicillin (50 IU/ml), streptomycin (50 RIg/ml), and eCG (Folligon: 2.5 IU/ml; Intervet, Boxmeer, Holland). The basic medium used for treatment of spermatozoa and fertilization of oocytes was essentially the same as that used by Parrish et al. [30] with minor modifications. The sperm preincubation medium (sperm TALP) consisted of Earle's Balanced Salt Solution (EBSS; Sigma) supplemented with gentamycin (50 Kig/ml), HEPES (25 mM), NaHCO 3 (25 mM), pyruvate (1 mM), lactate (10 mM), heparin (25 Rig/ml), caffeine (1 mM), hypotaurine (10 iM), MgCl 2 (25 jig/ml), and BSA (3 mg/ml; fraction V; Sigma). The fertilization medium (in vitro fertilization [IVF] TALP) consisted of EBSS supplemented with gentamycin (50 jig/ml), NaHCO 3 (25 mM), pyruvate (0.2 mM), lactate (10 mM), heparin (25 jig/ml), caffeine (1 mM), hypotaurine (10 jiM), and BSA (3 mg/ml). The embryo culture medium (ECM) was similar to that described by Fray et al. [31] and consisted of TCM-199 (with Earle's salts and sodium bicarbonate; Sigma) supplemented with 10% (v:v) heat-inactivated calf serum (Sigma), L-glutamine (0.4 mM), pyruvate (0.2 mM), lactate (10 mM), penicillin (50 IU/ml), and streptomycin (50 Rig/ml). Granulosa Cell Preparation and Culture Cryopreserved granulosa cells obtained from ovaries of slaughtered cows were used for the cell coculture. Granulosa cells were retrieved from batches of pooled bFF (-20 ml) obtained at the time of oocyte collection (see below) by centrifugation at 800 x g for 5 min. The cell pellet was suspended in 5 ml of PBS, and 10 ml of distilled water was added in order to lyse the erythrocytes that were usually present in the aspirated bFF After a brief 10-sec incubation, the same volume of PBS (triple-strength) was added to restore isotonicity. The cell suspension was then washed once with WM and once with ECM by centrifugation (800 g for 5 min each time). The granulosa cells were suspended in ECM and assessed for viability using the Trypan Blue dye exclusion method. For cryopreservation, the granulosa cells were pelleted and resuspended (6 x 106 cells/ml) in ECM plus 10% dimethyl sulfoxide (DMSO) and an extra 10% calf serum, then cooled to -70C (approximately - 1°C/min) before being plunged into liquid nitrogen. When required, frozen granulosa cells were thawed at 37C and washed twice in WM and ECM. For the coculture, 20 Il of granulosa cell suspension at a concentration of 2 105 cells/ml was placed in 30-Rl drops of ECM under oil, and cultured at 38.5°C in a humidified atmosphere of 5% CO 2. The medium was replenished every two days (i.e., 50% of drop volume replaced with fresh medium), and the cell monolayers were used for embryo coculture after 1 wk. Oocyte Maturation Bovine ovaries were collected from an abattoir and transported to the laboratory in a sterile saline solution supplemented with 1% (v:v) antibiotic and antimycotic solution (Sigma; cat. No. A9909). COCs were obtained by aspiration of 2- to 10-mm follicles with a 5-ml syringe attached to a 19-gauge needle. Only COCs with a complete corona layer and one or more compact cumulus cell layers were used. COCs were washed twice in WM and once in maturation media. Groups of 20 oocytes were randomly

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allocated into different treatment groups and cultured in 60Il droplets of MM under oil for 22-24 h, at 38.5°C in a humidified atmosphere of 5% CO 2. IVF and Embryo Culture For capacitation of the sperm, two straws of frozen bull semen from a single ejaculate were thawed in a water bath at 37°C for 20 sec. Spermatozoa were washed 4 times by centrifugation (800 g for 5 min each time) in sperm TALP medium and once in the IVF-TALP medium. The sperm pellet was then suspended in IVF-TALP medium at a concentration of 2 106 spermatozoa/ml before being placed into the IVF drops (final concentration: 1 X 106 spermatozoa/ml). Groups of approximately 10 oocytes were transferred to 20-Rl fertilization drops of IVF-TALP medium, under oil. A 30-,ul aliquot of the sperm suspension was introduced into the fertilization drops. The oocytes/sperm were incubated at 38.5°C in a humidified atmosphere of 5% CO 2. After 22-24 h, the presumptive embryos were washed in ECM and placed in coculture drops (20 per drop) containing granulosa cells. Three days after fertilization (fertilization = Day 0), the embryos were assessed for development, and the 4- to 16-cell embryos were transferred to new coculture drops. Thereafter, medium was replenished every two days (i.e., 50% of drop volume replaced with fresh medium), and embryos were examined again on Days 7 and 10 to assess their development. The number of embryos reaching the - 8-cell stage was recorded on Day 7 while the number forming blastocysts was recorded on Day 10. Experiments Depending on the number of good quality COCs recovered from each batch of ovaries, 1-3 culture drops were allocated per treatment with 20 COCs per drop. In each culture, equal numbers of drops were allocated to controls and treatment groups to avoid the introduction of bias into the cumulative data from a series of cultures. Note that in each experiment, treatments were present only during the 22-24-h oocyte maturation period before fertilization. Experiment 1. COCs: Effects of activin and follistatin. The dose-dependent effects of human recombinant (hr) activin-A and hr follistatin on COC maturation were studied. Activin-A was added to MM at concentrations of 0, 0.1, and 0.5 ,ug/ml. For follistatin, the doses tested were 0, 0.1, 1, and 10 g/ml. Experiment 2. COCs: Interaction between activin and follistatin. To test whether follistatin could reverse the effect of activin on COC maturation, a combination of activin and follistatin at different ratios was tested. Treatments were as follows: 1) MM only (control); 2) MM + activin-A (0.5 RIg/ml); 3) MM + follistatin (0.5 g/ml); 4) MM + follistatin (5 g/ml); 5) MM + activin-A (0.5 Rg/ml) + follistatin (0.5 jig/ml); 6) MM + activin-A (0.5 jig/ml) + follistatin (5 RIg/ml). Endogenous production of activin-A and follistatin during COC maturation was also measured in order to correlate with oocyte developmental potential and also to determine whether exogenous activin affected secretion of endogenous follistatin and vice versa. After COC maturation, 40 tl of medium (COC-conditioned medium) was collected from drops and frozen for hormone assay. Experiment 3. Cumulus-free oocytes. To assess any direct effects of activin-A and/or follistatin on oocytes, cu-

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SILVA AND KNIGHT

TABLE 1. Effect of activin-A present during IVM of bovine COCs on embryonic development (mean + SEM). No. oocytes cultured (n*)

-2-Cell

Control

457 (24)

61 ± 2

35 ± 2

13

1

56

3

21

2a

Activin (0.1 plg/ml)

326 (17)

62

3

35

2

15 ±

1ab

57

3

24

2 ab

Activin (0.5 jIg/ml)

445 (24)

63 +-2

37

2

18 ± 1b

Culture treatment

% Oocytes developing to 28-Cell Blastocyst

% Cleaved oocytes developing to -8-Cell Blastocyst

59 ± 3

29 ± 2 b

* Number of replicate culture drops. a,bDifferent superscripts within column indicate significant (p < 0.05) differences between treatment groups.

mulus cells were removed from good-quality COCs (with a complete corona layer and one or more compact cumulus layers). COCs were stripped of their cumulus cells by incubation (2 min at 37C) with hyaluronidase (1 mg/ml; Sigma) in WM and vortexing [32]. Oocytes that retained one or more corona cell layers were discarded. Only oocytes completely denuded or those with exposed zona pellucidae and just a few corona cells attached were used (hereafter referred to as cumulus-free oocytes). These oocytes were washed twice in WM and MM and were randomly allocated to each treatment group. Treatments were the same as in experiment 2. For all experiments, culture drops were examined 4 days postinsemination (i.e., 3 days after the beginning of coculture with granulosa cells) to assess the proportion of cleaved oocytes (> 2-cell stage). Development to the 8cell stage (recorded on Day 7) and blastocyst stage (recorded on Day 10) was expressed both as a proportion of total oocytes cultured and as a proportion of cleaved oocytes to adjust for differences in frequency of fertilization.

Statistical Analysis

Activin-A and Follistatin Assays

The results presented are based on experiments completed over an 18-mo period and involved more than 7000 individual COCs obtained from 26 batches of ovaries.

The proportions of both total oocytes and cleaved oocytes reaching defined developmental stages were transformed using the Freeman and Tukey transformation [34] before statistical analysis; for clarity, they are presented in tables as percentages ( SEM). Effects of each hormone treatment (different doses of activin and/or follistatin) were compared and analyzed by one-way analyses of variance of cumulative data from a minimum of five different batches of ovaries and > 6 replicate drops (120 oocytes) per treatment. When a significant F ratio was obtained, a posthoc protected least-squares difference test was used to compare individual means. A p value < 0.05 was considered to be significant. Linear regression analysis was used to examine the relationship between endogenous production of activin-A or follistatin by each group of 20 COCs and the proportion of cleaved oocytes developing to blastocysts. RESULTS

Total activin-A (i.e., free activin plus follistatin-bound activin) concentrations in COC-conditioned media were determined using a two-site ELISA described by Knight et al. [27]. Human recombinant (hr) activin-A (Genentech Inc., San Francisco, CA) was used as the standard and gave a dilution curve parallel to those for COC-conditioned media, bovine granulosa cell-conditioned culture media, and bFE Sensitivity was -100 pg/ml, and the intraassay and interassay coefficients of variation were 9.2% and 16.4%, respectively (n = 8 assays). Follistatin was determined by a novel in-house ELISA [33]. Pooled bFF was used as the working standard, since hr-follistatin gave a dilution curve steeper than those for bFF and COC-conditioned medium. One milliliter of bFF working standard was found to be equivalent to 3.93 jig of hr-follistatin [33]. Consequently, follistatin concentrations are expressed here in terms of the widely available hr-follistatin preparation provided by NIDDK (Bethesda, MD). The sensitivity was equivalent to -100 pg/ml. The intraassay and interassay coefficients of variation calculated from 6 assays were 5.3% and 14%, respectively. TABLE 2.

Experiment 1. COCs: Effects of Activin and Follistatin Oocyte cleavage rate (-60%) was not significantly affected by the addition of activin-A or follistatin to MM (Tables 1 and 2). Addition of 0.5 g/ml of activin-A to COC MM resulted in a 38% (p < 0.05) increase in the proportions of both total oocytes and cleaved oocytes reaching the blastocyst stage (Table 1). The lower dose of activin-A (0.1 pIg/ml) promoted a 14% increase, but this effect was not statistically significant. On the other hand, the addition of follistatin to COC MM decreased the proportions of total oocytes and of cleaved oocytes reaching the blastocyst stage. This effect was dose-dependent and significant at doses of 1 and 10 ,ug/ml (Table 2). When 10 tug/ml of follistatin was present during COC maturation, the proportion of blastocysts produced after IVF was reduced by 67%. Follistatin also reduced the proportion of embryos developing to the 8-cell stage in a dose-dependent way, although not so dramati-

Effect of follistatin present during IVM of bovine COCs on embryonic development (mean + SEM).

Culture treatment

No. oocytes cultured (n*)

% Oocytes developing to -2-Cell

-8-Cell

Control

115 (6)

60

5

33

Follistatin (0.1 pig/ml) Follistatin (1 g/ml) Follistatin (10 ig/ml)

119 (6) 115 (6) 118 (6)

57 + 2 60 4 3 54

30 27 23

14

3 +

2 4

% Cleaved oocytes developing to Blastocyst

b b

b

2

2

a

10 2 b 7 2 bc 4 -+ 1

* Number of replicate culture drops. a,b,c Different superscripts within column indicate significant (p < 0.05) differences between treatment groups.

-8-Cell

Blastocyst

55

4

24

53 44 42

5ab

18 3ab 11 ± 2 bc 1 8

6ab b

3

3a

ACTIVIN, FOLLISTATIN, AND BOVINE OOCYTE MATURATION TABLE 3.

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Effects of activin-A (Act) and follistatin (FS) present during IVM of bovine COCs on embryonic development (mean ± SEM). No. oocytes

Culture treatment

cultured (n*)

Control Act (0.5 g/ml) FS (0.5 g/ml) FS (5 g/ml) Act (0.5 Rg/ml) + FS (0.5 [Lg/ml) Act (0.5 g/ml) + FS (5 g/ml)

480 465 449 470 477 495

(25) (25) (25) (25) (25) (25)

0%

Oocytes developing to

-2-Cell

-8-Cell

59 61 58 54 61 57

32 + 2, b 35 + 2 2 9 ± 3b 22 ± 3c 34 + 2 b 29 + 21b

+ 2a + 2 + 3a + 3a + 3a + 2a

% Cleaved oocytes developing to Blastocyst 13 + 1a 16 ± 1 8 ± 2b 5 l1b 14 _ 2a 7 + 1b

Ž8-Cell 54 57 48 40 55 52

+ 3a + 3 + 3a + 4b + 3a + 3a

Blastocyst 21 27 14 10 22 13

+ 2a 2b + 3 + 2c ± 2a ± 2c

Number of replicate culture drops. a,b,cDifferent superscripts within column indicate significant (p < 0.05) differences between treatment groups. *

cally as the reduction in the proportion of blastocysts, and statistically significant only at the highest dose.

(a)

r=0.57; p8 m

10

1000

100

Activin (ng/ml)

(b)

r=0.36; p