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FSH-stimulated follicular secretions enhance oocyte maturation in pigs
Theriogeno/ogy41:1473-1481,1994
FSH-STIMULATED FOLLICULAR SECRETIONS ENHANCE OOCYTE MATURATION IN PIGS J. Dinga.md G.R. Foxcroftb Department of Animal Science, Faculty of Agriculture and Forestry University of Alberta, Edmonton, Alberta, Canada T6G 2P5 Received for publication: Accepted
October 5, 1993 March I, 1994
ABSTRACT Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theta and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 pg/ml FSH (FSHstimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2Spg/ml FSH, 2.5 @ml LH and 20 @ml PRL, FSH-LH-PRLstimulated conditioned medium; 54.1 vs 28.5%; P=O.OOl). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=O.O6). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=O.65), but were higher than those of oocytes matured in fresh control medium (PcO.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment. Key words: pig, follicle, conditioned
medium, oocyte, maturation
Acknowledgments This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Alberta Pork Producers Development Corporation. The authors thank Mingxian Shen and Dorothy Payne for their help in IVM-IVF and RIA, respectively. We also thank Alberta Swine AI Center for supplying boar semen; the National Institute of Diabetes and Digestive and Kidney Diseases for gifts of gonadotropins. aPresent address: Animal Biotechnology Embryo Laboratory, Department of Biomedical Sciences, University of Guelph, Ciuelph, Ontario, Canada NlG 2Wl. bcorrespondence and reprint requests.
Copyright
0 1994 Butterworth-Heinemann
Theriogenology
1474 INTRODUCTION
Follicular cell co-culture supports both nuclear (Ding et al., unpublished observations) and cytoplasmic (4, lo- 11, 13- 14, 20) maturation in porcine oocytes. Furthermore, it has been reported that supplementation of oocyte maturation medium with porcine follicular fluid (7, 15 16, 19) or use of follicle conditioned medium (10-l 1) to culture immature pig oocytes in vitro increased the rate of male pronuclear formation and developmental capacity after in vitro fertilization. These results, therefore, suggest that factors secreted by follicular cells are involved in regulating oocyte maturation. Gonadotropins are known to affect follicular cell differentiation. During the follicular phase in vivo, FSH induces early maturational changes and stimulates estradiol production by follicular cells, resulting in the release of the preovulatory surge of LH. The LH (and probably also PRL) induces luteinization of follicular cells and ovulation and stimulates progesterone production (8). This series of maturational changes is also paralleled by a complex series of events determining follicle-oocyte interactions and hence oocyte maturation (6). Therefore, follicular cell conditioned media produced in the presence of different gonadotropin combinations may contain factors secreted by follicular cells which in both quantitative and qualitative terms may differentially affect oocyte maturation. Furthermore, since follicular cells may undergo maturational differentiation during the course of in vitro culture, conditioned media collected over different periods may vary in their ability to support oocyte maturation. The objectives of the present study were to test the above hypotheses and to set up a maturation system using conditioned medium for studies of pig oocytes. MATERIALS
AND METHODS
Preparation of Oocytes and Follicular Shells Ovaries were collected from slaughtered prepubertal gilts of about 100 kg liveweight, at a local abattoir and transported to the laboratory in a polystyrene box to prevent major changes in temperature. Follicles with a diameter of 3 to 7 mm were dissected from the ovaries, and oocyte-cumulus-mural granulosa cell complexes (oocyte-complexes) were isolated from healthy follicles and were used as the source of immature oocytes. Follicular shells from 5 to 7-mm healthy follicles with oocytes and follicular fluid removed were used to produce conditioned media. All dissection procedures have been described previously (4). Production of Follicular Shell Conditioned
Media and Gocyte Maturation
Experiment 1. Four batches of follicular conditioned media were produced. In each batch, 5 follicular shells were cultured in 10 ml oocyte culture medium (2 ml/shell) containing 2Spg/ml FSH (USDA-pFSH-B-l,AFP-5600) only (FSH-stimulated conditioned medium); another 5 follicular shells were cultured in 10 ml of culture medium containing 2.5 @ml FSH, 2.5 pg/ml LH (NIADDK-oLH-26, AFP-5551b) and 20 @ml PRL (USDA-pprl-B-l, AFP-5000), a hormone combination previously used for oocyte maturation (FSH-LH-PRL-stimulated conditioned medium; 4). The oocyte maturation medium was TCM199 (with Earles’ salts and L-glutamine, Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (PCS, Gibco, Grand Island,
Theriogenology
1475
NY); 100 pg/ml glutamine (BDH, Toronto, Canada); 70 @ml L-ascorbic acid (BDH, Toronto, Canada); and 35 pg/ml insulin (Sigma, St Louis, MO). Culture was carried out in a 50-ml (25 cm’) tissue culture flask (Falcon 3103) on a rocking platform in 5% CO2 in humidified air at 39’C. After 48 h of culture, the conditioned media were centrifuged at 2083 x g for 10 min, and the supernatants were filtered using a 0.22~pm micro-syringe filter and preserved at -30°C until use. A randomized complete block experiment was designed to examine the effect of follicular conditioned media on oocyte maturation. The experiment was replicated 5 times using 4 batches of conditioned media, and immature oocyte-complexes used in each replicated trial were randomly divided into 6 culture dishes (10 to 15 oocytes per dish) and subjected to 6 different culture conditions: oocytes cultured in 2 ml FSH-stimulated conditioned medium without or with further supplementation of gonadotropins (2.5 pg/ml FSH, 2.5 pg/ml LH and 20 ngfml PRL, Treatment 1 and 2, respectively); in FSH-LH-PRL-stimulated conditioned medium without and with further supplementation of gonadotropins (Treatments 3 and 4, respectively); in fresh culture medium with supplementation of gonadotropins (Treatment 5, Control I); and in fresh culture medium supplemented with gonadotropins and one follicle shell (Treatment 6, Control II). Gocytes were cultured for 47 h using a rocking system under the same gas and temperature conditions as used for the production of conditioned media. Exneriment 2. In the first experiment it was shown that follicular secretions stimulated by FSH during the production of conditioned medium support cytoplasmic maturation of oocytes. In Experiment 2 we therefore tested the effects of follicular secretions produced during the different periods of conditioning under the stimulation of FSH. The 48-h conditioning period was divided into 2 consecutive 24-h periods, and because the conditioning period was halved the number of follicular shells was doubled. To ensure an adequate supply of the conditioned media, 7 batches of follicular conditioned media were produced by culturing 10 follicular shells in 10 ml oocyte culture medium (1 ml/shell) supplemented with 2.5 pg/ml FSH. After 24 h of culture, the medium was collected and replaced with 10 ml fresh medium supplemented with 2.5pgAnl FSH, followed by culture for another 24 h. The medium was then collected again. The conditioned media were centrifuged, filtered and then stored until use as described in Experiment 1. A randomized, complete block design was used again for the oocyte maturation trial. The experiment consisted 3 treatment groups: 1) oocyte-complexes cultured in FSH-stimulated follicular conditioned medium produced during the first 24 h, 2) cultured in FSH-stimulated follicular conditioned medium produced during the second 24 h, and 3) cultured in fresh culture medium (control). Gonadotropins (2.5 pg/ml FSH, 2.5 &ml LH and 20 nglml PBL) were added to each treatment during the oocyte maturation period. The experiment was replicated 4 times using 4 of the 7 batches of conditioned media produced and duplicate oocyte cultures for each treatment. Fertilization
In Vitro
After 47 h in culture, the cumulus-enclosed oocytes from each culture dish were transferred to 1 ml fertilization medium (BO medium; 2) containing BSA (fraction V; 10 mg/ml)
Theriogenology
1476
and 2 mM caffeine (all chemicals were from Sigma or BDH, Toronto, Canada) and were inseminated with 5 x 10s in vitro capacitated boar spermatozoa. The procedures for in vitro capacitation of freshly ejaculated boar spermatozoa were as described previously (3-4). After 6 h of co-culture with spermatozoa, the oocytes were transferred into 1 ml sperm-free modified Krebs’Ringer-bicarbonate medium (Table 1; 17), cultured for another 5 h and then prepared for evaluation of nuclear status. Table 1. Composition
of the modified Krebs’-Ringer-bicarbonate mg/lOO ml
Components
NaCl 670.5 KC1 35.6 CaC1,.2H.h,0 24.5 MgS0,.7H,O 29.4 NaHCO, 210.6 D-Glucose 100 Glutamine 14.6 400 BSA (fraction V) Kanamycin 10 Deionized water was added to 1OOml Osmolarity was adjusted to 290 mOs
medium mM 114.7 4.78 1.70 1.19 25.07 5.55 1.00
Evaluation of Nuclear Status Gocytes were denuded of cumulus cells, mounted on a slide with a whole-mount technique, and fixed for at least 48 h in 25% acetic acid in ethanol. Oocytes were then stained with 1% lacmoid in 45% acetic acid and their nuclear status examined under a phase-contrast microscope. Oocytes were identified as non-penetrated oocytes with GV, PMI, MI, MI1 and penetrated oocytes with detached unswollen or slightly swollen sperm head(s) (SHl), with further swollen sperm head(s), and/or with male pronucleus/pronuclei, as described previously (4-5). Steroid Measurements
of Conditioned Medium
Progesterone and estradiol in conditioned medium were determined by direct (none extraction) RIA, as described previously (4). Standard curves for progesterone and estradiol-17B ranged from 0.00313 to 6.40 and 0.0025 to 0.80 ng per tube, respectively. Sensitivities of the assays, defined as the dose at binding (%> = CB, - 2SDB,,) x 100% / CB_ (CB,,: mean cpm of maximal binding, SDB,,: standard deviation of cpm of maximal binding tubes), were 6.25 and 1.56 pg per tube for progesterone and estradiol, respectively. To remove any effects of interassay variability on treatment differences, all samples were assayed in a single assay with intraassay CVs of 5.31 and 6.48% for progesterone and estradiol-17R, respectively.
1477
Theriogenology
Statistical Analysis Data were analyzed using ANOVA for a randomized, complete block design (replicated trials were blocks; Model Y = block + treatment + error, 18). Percentage data were subjected to arcsine transformation before analysis. Statistical analysis was carried out using the general linear model procedures of the Statistical Analysis System (Version 6.07, SAS Institute, Cary, NC). Multiple comparisons were made by the PDIFF (LSD) function of SAS. Orthogonal contrast analysis was also used to analyze main effects in Experiment 1 (FSH-stimulated vs FSH-LHPRL-stimulated conditioned media; further addition of FSH-LH-PRL during oocyte maturation vs no FSH-LH-PRL addition; 18). RESULTS Experiment
1
Table 2 shows the effect of follicular conditioned media on oocyte maturation. The proportion of oocytes developing beyond the second metaphase (>MII) and the penetrability of MIl oocytes were not affected by supplementation treatment. Male pronuclear development in penetrated oocytes, however, was significantly affected by treatment (Table 2). Multiple comparisons showed that the high rate of male pronuclear formation in penetrated oocytes matured in FSH-stimulated follicular conditioned medium supplemented with the FSH-LH-PRL mixture during maturation culture (Treatment 2) was similar to that in oocytes matured in fresh maturation medium co-cultured with one follicular shell (Treatment 6, P=O.82), but was significantly higher than in oocytes matured in fresh maturation medium without co-culture with a follicular shell (Treatment 5; P=O.O5). Oocytes matured in FSH-LH-PRL-stimulated follicular conditioned medium (Treatments 3 and 4) had lowest male pronuclear formation rates. Orthogonal contrasts also showed that oocytes matured in FSH-stimulated follicular conditioned medium (Treatments 1 and 2) had a significantly higher male pronuclear formation rate than those oocytes matured in FSH-LI-I-PRL-stimulated follicular conditioned medium (Treatments 3 and 4, P=O.O.OOl). Furthermore, addition of the FSH-LH-PRL mixture during the oocyte maturation period may increase the male pronuclear formation rate (comparison of Treatments 2 and 4 with Treatments 1 and 3; P=O.O6). With respect to the steroid content of conditioned media neither estradiol concentrations [13.76*2.98 (n=4) and 18.75f2.47 (n=4) q/ml, respectively] nor progesterone concentrations [537.46&136.3 (n=4) and 864.6f346.0 (n=4) @ml, respectively] in FSH- and FSH-LH-PRLstimulated follicular conditioned media were significantly different (lXt.19). Experiment
2
Table 3 shows that the treatments did not alter nuclear maturation rates (Pa.05) or penetrability (PM.05) of in vitro matured oocytes. However, male pronuclear formation rate was higher in oocytes matured in both conditioned media than in those matured in fresh culture medium (PcO.05). Male pronuclear formation rates were similar in both the first 24-h and the second 24-h groups, and were similar to those matured in FSH-stimulated follicular conditioned medium supplemented with FSH-LH-PRL during oocyte maturation in Experiment 1.
1478
Theriogenology
Table 2. Effect of follicular shell conditioned medium on oocyte nuclear maturation (WI), sperm penetration and male pronuclear development (MPN)
Treatments
Culture medium
1
FSH-stimulated
FSH-LH-PRL addition Number of oocytes (mean%~SEI@ during Examined oocyte >MII penetrated with MPN maturation -
63 (93.Z.2)d
+
2
3
FSH-LH-PRLstimulated
+
4
5
6
-
Fresh medium
Fresh medium + IFS
+
+
62
68
(%.E3A)d
58 (%.c+4.O)d
(7LZ9.7)d
62 (92.3*5.7)6
60 (97.5f2.qd
(28.iz7.7)’
(94.Z2.5)d
(93.::6.7,”
(40.:8.6)c1
64 (98.8f1.2)’
(95Z4.5)d
(4Lz8.4)d
(92.32.O)d
59 (98.5f1.5)d
(71.42615.O)d
62
65
64
’ The experiment was replicated for five times. b Mean percentage of >MII oocytes: mean of the percentage of the total number of oocytes examined that developed beyond MII; Mean percentage of penetrated oocytes: mean of the percentage of all >MII oocytes penetrated by spermatozoa; and Mean percentage of IWN oocytes: mean of the percentage of all penetrated MI1 oocytes with MPN. ’ One foIlicuIar shell was co-cultured with oocytes. d-fMeans within a column without common superscripts were significantly different (P < 0.05).
The progesterone concentration of 179.05i21.3 q/ml (n=6) in the first 24-h conditioned medium was significantly lower than that of 307.51f50.22 rig/ml (n=7) in the second 24-h conditioned medium (P=O.O46). Conversely, the estradiol concentration of 15.76f1.66 ng/ml (n=7) in the first 24-h conditioned medium was higher than that of 10.48f1.78 ng/ml (n=6) in the second 24-h conditioned medium (P=O.O43). DISCUSSION Follicular secretions obtained from culture of follicular shells under the stimulation of FSH, LH and PRL have been reported to enhance cytoplasmic maturation in the porcine
1479
Theriogenology
Table 3. Effects of follicular secretions produced during the different culture periods under the stimulation of FSH on oocyte maturation @III), sperm penetration and male pronuclear development (MPN).
Treatment groups
No. replicates
No. oocytes examined
No. oocytes >MII (mean%*seY
No. oocytes penetrated (mean%*seY
No. oocytes With MPN (mean%fse)
1) First 24-h*
8
75
72 (95.2f2.6)”
56 (79.9f8.5)’
36 (61.51t5.8)”
2) Second 24-hd
8
86
73 (85.3f3.9)
(83. p:.,)
40 (66.0f9.0)
(95.c3.8)
(88.87z4.8)
(37.Z9.3)’
3) Fresh Medium
8
84
’ Mean of the percentage of the total number of oocytes developed beyond the MI1 stage (>MII). b Mean of the percentage of all >MII oocytes penetrated by spermatozoa. ’ Mean of the percentage of all penetrated MII oocytes with MPN. * First 24-h: conditioned medium collected after 24 h of culture of 10 follicular shells in 10 ml of oocyte maturation medium supplemented with 2.5 pgknl FSH, Second 24-h: after collection of the first 24-h conditioned medium, the follicular shells were cultured for a second 24 h in 10 ml of fresh oocyte maturation medium containing 2.5 &ml FSH and then conditioned medium was harvested. e-l Means within a column without common superscripts were significantly different (P < 0.05).
oocyte when compared with the control medium (10-l 1). Our results, however, did not confirm this effect. The present results show that follicular secretions stimulated by FSH alone supported cytoplasmic maturation more effectively than follicular secretions stimulated by a combination of FSH, LH and PRL, and which was comparable to the results obtained from co-culture with follicular shells. If anything, follicular secretions stimulated by a combination of FSH, LH and PRL (similar to the treatment of Mattioli et al. 10-l 1) exerted an inhibitory effect on cytoplasmic maturation when compared with fresh culture medium. The precise reasons for this are not clear. However, several differences exist between the 2 culture systems. In our study the ocytes were fixed 10 to 11 h after insemination, while in the above studies (10-l 1) they were fixed at 14 h. Cumulus-enclosed oocytes associated with a piece of mural granulosa were used as a source of immature oocytes in our culture, while cumulus-enclosed oocytes were used by Mattioli et al. (10-l 1). Follicular tissue and oocytes used in our cultures were obtained from prepubertal gilts, as compared with cyclic gilts. Finally, follicular conditioned media in our study were produced by culturing follicular shells alone; Mattioli et al. (10-11) produced conditioned media in the presence of oocytes, and factors resulting from interactions between follicular cells and oocytes were therefore probably involved.
1480
Theriogenology
The reason for FSH-stimulated follicular secretions promoting cytoplasmic maturation is not known. Physiologically, FSH effects are dominant before the time of the LH surge. Granulosa and theta cells stimulated by FSH at this stage are able to convert androgens of thecal origin to estrogen, resulting in an estrogen-dominated environment in the follicle. After the LH surge, steroid production is substantially altered. Production of estrogen declines and production of progesterone increases (1, 8, 9). Estradiol and progesterone production by follicular shells stimulated by FSH alone or by a combination of FSH, LH and PRL in our present study were not significantly different (P > 0.05), although there was a trend for follicular shells stimulated by FSH alone to be. less steroidogenic than when stimulated by a mixture of FSH, LH and PRL (estradiol: 13.76k2.98 to 18.75k2.47; progesterone: 537.46f136.3 to 864.6f346.0 ng/ml). It seems that follicular cells stimulated by FSH alone also underwent luteinization during culture, which is not comparable to the in vivo situation. This is probably caused by LH contamination (~1%) in FSH (USDA-pFSH-B-1, AFP-5600). The overall change in the progesterone to estradiol ratio in both conditioned media were similar to those found in follicular fluid just before ovulation (100 to 108 h/30 to 36 h post eCG/hCG; 1). Mattioli et al. (11) showed that steroid hormones (progesterone in particular) are mainly responsible for the improvement in cytoplasmic maturation of oocytes matured in conditioned medium. Our results do not imply this. Rather, they further support earlier suggestions that nonsteroidal follicular factors are involved in the regulation of oocyte cytoplasmic maturation (4). Further supplementation of a mixture of FSH, LH and PRL with both types of conditioned media during oocyte culture marginally improved the degree of cytoplasmic maturation of oocytes (P=O.O6, Table 2). It has been reported by Mattioli et al. (12) that oocytes matured in medium containing FSH only had a low male pronuclear formation rate (440/o), while those matured in medium containing LH had a high male pronuclear formation rate (72%). Thus the effect of further supplementation of gonadotropins during oocyte maturation, in conditioned medium stimulated by FSH alone, may be explained by an LH effect on cytoplasmic maturation. However, this would not explain an effect in oocytes matured in conditioned medium stimulated by a mixture of FSH, LH and PRL. One possibility is that the hormones had been used up by follicular cells during the follicle conditioning period. Conditioned medium produced in the first or second 24-h period of culture in the presence of FSH had a similar effect on oocyte nuclear and cytoplasmic maturation, suggesting that the concentrations of stimulating factors in these 2 conditioned media am similar, although the degree of luteinization of follicular cells was lower in the first 24-h period than in the second 24-h period of culture. This suggests that steroidal factors may not be the most critical regulators of cytoplasmic maturation. In summary, the results of our study suggest that FSH-stimulated highly suitable for supporting porcine oocyte maturation in vitro.
follicular secretions are
1481
Theriogenology REFERENCES
1. Ainsworth L, Tsang BK, Downey BR, Marcus GM and Amstrong DT. Interrelationships between follicular fluid steroid levels, gonadotropic stimuli, and oocyte maturation during preovulatory development of porcine follicles. Biol Reprod 1980; 23:621-627. 2. Brackett BG and Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975; 12260-274. 3. Ding J, Clarke N, Nagai T and Moor RM. Protein and nuclear changes in pig eggs at fertilization. Mol Reprod Dev 1992; 31:287-296. 4. Ding J and Foxcroft GR. Follicular heterogeneity and oocyte maturation in via0 in pigs. Bio Reprod 1992; 47648-655. 5. Ding J, Moor R&l and Foxcroft GR. Effect of protein synthesis on maturation, sperm penetration, and pronuclear development in porcine oocytes. Mol Reprod Dev 1992; 3359-66. 6. Foxcroft GR, Cosgrove JR, Ding .I, Hofacker S and Wiesak T. Reproductive function: current concepts. In: Cole DJA, Wiseman J and Varley MA (eds), Principles of Pig Science Nottingham University Press, 1993; In press 7. Funahashi H and Day BN. Effects of different serum supplements in maturation medium on meiotic and cytoplasmic maturation of pig oocytes. Theriogenology 1993; 39965-973. 8. Gore-Langton RE and Armstrong DT. Follicular steroidogenesis and its control. In: Knobil E, Neil1 J, Ewing LL, Markert CL, Greeneald GS and Pfaff DW (eds), The Physiology of Reproduction. New York, Raven Press, 1988; 331-385. 9. Grant SA, Hunter MG and Foxcroft GR. Morphological and biochemical characteristics during ovarian follicular development in the pig. J Reprod Fertil 1989; 86171-183. 10. Mattioli M, Galeati G and Seren E. Effect of follicle somatic cells during pig oocyte maturation on egg penetration and male pronuclear formation. Gamete Res 1988; 20:177-183. 11. Mattioli M, Galeati G, Bacci ML and Seren E. Follicular factors influence cocyte fenilizability by modulating the intercellular co-operation between cumulus cells and oocytes. Gamete Res 1988; 21:223-232. 12. Mattioli M, Bacci ML, Galeati G and Seren E. Effects of LH and FSH on the maturation of pig oocytes in vitro. Theriogenology 1991; 36:95-105. 13. Moor RM, Mattioli M, Ding J and Nagai T. Maturation of pig occytes in viva and in vitro. J Reprod Fertil Suppl 1990; 40:197-210. 14. Nagai T, Ding J and Moor RM. Effect of follicular cells and steroidogenesis on maturation and fertilization in vitro of pig oocytes. J Exp Zoo1 1993; 266146-151. 15. Naito K, Fukuda Y and Toyoda Y. Effects of porcine follicular fluid on male pronuclear formation in porcine oocytes maNed in vitro. Gamete Res 1988; 21:289-295. 16. Naito K, Fukuda Y and Ishibashi I. Developmental ability of porcine ova matured in porcine follicular fluid in vitro and fertilized in vitro. Theriogenology 1989; 31:1049-1057. 17. Petters RM, Johnson BH, Reed ML and Archibong AE. Glucose, glutamine and inorganic phosphate in early development of the pig embryo in vitro. J Reprod Fettil 1990, 89:269-275. 18. Steel RGD and Torrie JH. Principles and Procedures of Statistics, A biometrical Approach. McGraw-Hill Book Company, 1980. 19. Yoshida M, Ishizaki Y, Kawagishi H, Bamba K and Kojima Y. Effect of pig follicular fluid on maturation of pig oocytes invitro and on their subsequent fertilizing and developmental capacity invitro. 1 Reprod Fertil 1992; 95:481-488. 20. Zheng YS and Sirard MA. The effect of sera, bovine semm albumin and folhcular cells on the in vitro maturation and fertilization of porcine oocytes. Theriogenology 1992; 37:779-790.
FSH-STIMULATED FOLLICULAR SECRETIONS ENHANCE OOCYTE MATURATION IN PIGS J. Dinga.md G.R. Foxcroftb Department of Animal Science, Faculty of Agriculture and Forestry University of Alberta, Edmonton, Alberta, Canada T6G 2P5 Received for publication: Accepted
October 5, 1993 March I, 1994
ABSTRACT Follicular secretions can support cytoplasmic maturation in vitro in the pig. The effects of follicular secretions stimulated in vitro by different combinations of gonadotropins and over different culture periods on cytoplasmic maturation of the pig oocyte were studied. In Experiment 1, follicular shells (including theta and mural granulosa cells) from 5 to 7-mm follicles were cultured in vitro under the stimulation of different combinations of gonadotropins for 48 h, and then the obtained conditioned media were used for oocyte maturation. Oocytes cultured in conditioned medium harvested after treatment of follicular shells with 2.5 pg/ml FSH (FSHstimulated conditioned medium) yielded a higher percentage of male pronuclear formation than those matured in conditioned medium harvested after culture of follicular shells with a combination of hormones (2Spg/ml FSH, 2.5 @ml LH and 20 @ml PRL, FSH-LH-PRLstimulated conditioned medium; 54.1 vs 28.5%; P=O.OOl). Addition of the combination of FSH, LH and PRL during the period of oocyte maturation marginally improved male pronuclear formation rates (41.3 vs 55.6%; P=O.O6). In Experment 2, follicular shells were cultured under the stimulation of FSH only. Conditioned media were harvested after the first 24 h and the second 24 h of culture. The rates of male pronuclear formation in oocytes matured in these 2 conditioned media did not differ (P=O.65), but were higher than those of oocytes matured in fresh control medium (PcO.03). It is concluded that factors secreted by follicular cells stimulated by FSH alone provide better support for full oocyte maturation in the pig than by combined FSH, LH and PRL treatment. Key words: pig, follicle, conditioned
medium, oocyte, maturation
Acknowledgments This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Alberta Pork Producers Development Corporation. The authors thank Mingxian Shen and Dorothy Payne for their help in IVM-IVF and RIA, respectively. We also thank Alberta Swine AI Center for supplying boar semen; the National Institute of Diabetes and Digestive and Kidney Diseases for gifts of gonadotropins. aPresent address: Animal Biotechnology Embryo Laboratory, Department of Biomedical Sciences, University of Guelph, Ciuelph, Ontario, Canada NlG 2Wl. bcorrespondence and reprint requests.
Copyright
0 1994 Butterworth-Heinemann
Theriogenology
1474 INTRODUCTION
Follicular cell co-culture supports both nuclear (Ding et al., unpublished observations) and cytoplasmic (4, lo- 11, 13- 14, 20) maturation in porcine oocytes. Furthermore, it has been reported that supplementation of oocyte maturation medium with porcine follicular fluid (7, 15 16, 19) or use of follicle conditioned medium (10-l 1) to culture immature pig oocytes in vitro increased the rate of male pronuclear formation and developmental capacity after in vitro fertilization. These results, therefore, suggest that factors secreted by follicular cells are involved in regulating oocyte maturation. Gonadotropins are known to affect follicular cell differentiation. During the follicular phase in vivo, FSH induces early maturational changes and stimulates estradiol production by follicular cells, resulting in the release of the preovulatory surge of LH. The LH (and probably also PRL) induces luteinization of follicular cells and ovulation and stimulates progesterone production (8). This series of maturational changes is also paralleled by a complex series of events determining follicle-oocyte interactions and hence oocyte maturation (6). Therefore, follicular cell conditioned media produced in the presence of different gonadotropin combinations may contain factors secreted by follicular cells which in both quantitative and qualitative terms may differentially affect oocyte maturation. Furthermore, since follicular cells may undergo maturational differentiation during the course of in vitro culture, conditioned media collected over different periods may vary in their ability to support oocyte maturation. The objectives of the present study were to test the above hypotheses and to set up a maturation system using conditioned medium for studies of pig oocytes. MATERIALS
AND METHODS
Preparation of Oocytes and Follicular Shells Ovaries were collected from slaughtered prepubertal gilts of about 100 kg liveweight, at a local abattoir and transported to the laboratory in a polystyrene box to prevent major changes in temperature. Follicles with a diameter of 3 to 7 mm were dissected from the ovaries, and oocyte-cumulus-mural granulosa cell complexes (oocyte-complexes) were isolated from healthy follicles and were used as the source of immature oocytes. Follicular shells from 5 to 7-mm healthy follicles with oocytes and follicular fluid removed were used to produce conditioned media. All dissection procedures have been described previously (4). Production of Follicular Shell Conditioned
Media and Gocyte Maturation
Experiment 1. Four batches of follicular conditioned media were produced. In each batch, 5 follicular shells were cultured in 10 ml oocyte culture medium (2 ml/shell) containing 2Spg/ml FSH (USDA-pFSH-B-l,AFP-5600) only (FSH-stimulated conditioned medium); another 5 follicular shells were cultured in 10 ml of culture medium containing 2.5 @ml FSH, 2.5 pg/ml LH (NIADDK-oLH-26, AFP-5551b) and 20 @ml PRL (USDA-pprl-B-l, AFP-5000), a hormone combination previously used for oocyte maturation (FSH-LH-PRL-stimulated conditioned medium; 4). The oocyte maturation medium was TCM199 (with Earles’ salts and L-glutamine, Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (PCS, Gibco, Grand Island,
Theriogenology
1475
NY); 100 pg/ml glutamine (BDH, Toronto, Canada); 70 @ml L-ascorbic acid (BDH, Toronto, Canada); and 35 pg/ml insulin (Sigma, St Louis, MO). Culture was carried out in a 50-ml (25 cm’) tissue culture flask (Falcon 3103) on a rocking platform in 5% CO2 in humidified air at 39’C. After 48 h of culture, the conditioned media were centrifuged at 2083 x g for 10 min, and the supernatants were filtered using a 0.22~pm micro-syringe filter and preserved at -30°C until use. A randomized complete block experiment was designed to examine the effect of follicular conditioned media on oocyte maturation. The experiment was replicated 5 times using 4 batches of conditioned media, and immature oocyte-complexes used in each replicated trial were randomly divided into 6 culture dishes (10 to 15 oocytes per dish) and subjected to 6 different culture conditions: oocytes cultured in 2 ml FSH-stimulated conditioned medium without or with further supplementation of gonadotropins (2.5 pg/ml FSH, 2.5 pg/ml LH and 20 ngfml PRL, Treatment 1 and 2, respectively); in FSH-LH-PRL-stimulated conditioned medium without and with further supplementation of gonadotropins (Treatments 3 and 4, respectively); in fresh culture medium with supplementation of gonadotropins (Treatment 5, Control I); and in fresh culture medium supplemented with gonadotropins and one follicle shell (Treatment 6, Control II). Gocytes were cultured for 47 h using a rocking system under the same gas and temperature conditions as used for the production of conditioned media. Exneriment 2. In the first experiment it was shown that follicular secretions stimulated by FSH during the production of conditioned medium support cytoplasmic maturation of oocytes. In Experiment 2 we therefore tested the effects of follicular secretions produced during the different periods of conditioning under the stimulation of FSH. The 48-h conditioning period was divided into 2 consecutive 24-h periods, and because the conditioning period was halved the number of follicular shells was doubled. To ensure an adequate supply of the conditioned media, 7 batches of follicular conditioned media were produced by culturing 10 follicular shells in 10 ml oocyte culture medium (1 ml/shell) supplemented with 2.5 pg/ml FSH. After 24 h of culture, the medium was collected and replaced with 10 ml fresh medium supplemented with 2.5pgAnl FSH, followed by culture for another 24 h. The medium was then collected again. The conditioned media were centrifuged, filtered and then stored until use as described in Experiment 1. A randomized, complete block design was used again for the oocyte maturation trial. The experiment consisted 3 treatment groups: 1) oocyte-complexes cultured in FSH-stimulated follicular conditioned medium produced during the first 24 h, 2) cultured in FSH-stimulated follicular conditioned medium produced during the second 24 h, and 3) cultured in fresh culture medium (control). Gonadotropins (2.5 pg/ml FSH, 2.5 &ml LH and 20 nglml PBL) were added to each treatment during the oocyte maturation period. The experiment was replicated 4 times using 4 of the 7 batches of conditioned media produced and duplicate oocyte cultures for each treatment. Fertilization
In Vitro
After 47 h in culture, the cumulus-enclosed oocytes from each culture dish were transferred to 1 ml fertilization medium (BO medium; 2) containing BSA (fraction V; 10 mg/ml)
Theriogenology
1476
and 2 mM caffeine (all chemicals were from Sigma or BDH, Toronto, Canada) and were inseminated with 5 x 10s in vitro capacitated boar spermatozoa. The procedures for in vitro capacitation of freshly ejaculated boar spermatozoa were as described previously (3-4). After 6 h of co-culture with spermatozoa, the oocytes were transferred into 1 ml sperm-free modified Krebs’Ringer-bicarbonate medium (Table 1; 17), cultured for another 5 h and then prepared for evaluation of nuclear status. Table 1. Composition
of the modified Krebs’-Ringer-bicarbonate mg/lOO ml
Components
NaCl 670.5 KC1 35.6 CaC1,.2H.h,0 24.5 MgS0,.7H,O 29.4 NaHCO, 210.6 D-Glucose 100 Glutamine 14.6 400 BSA (fraction V) Kanamycin 10 Deionized water was added to 1OOml Osmolarity was adjusted to 290 mOs
medium mM 114.7 4.78 1.70 1.19 25.07 5.55 1.00
Evaluation of Nuclear Status Gocytes were denuded of cumulus cells, mounted on a slide with a whole-mount technique, and fixed for at least 48 h in 25% acetic acid in ethanol. Oocytes were then stained with 1% lacmoid in 45% acetic acid and their nuclear status examined under a phase-contrast microscope. Oocytes were identified as non-penetrated oocytes with GV, PMI, MI, MI1 and penetrated oocytes with detached unswollen or slightly swollen sperm head(s) (SHl), with further swollen sperm head(s), and/or with male pronucleus/pronuclei, as described previously (4-5). Steroid Measurements
of Conditioned Medium
Progesterone and estradiol in conditioned medium were determined by direct (none extraction) RIA, as described previously (4). Standard curves for progesterone and estradiol-17B ranged from 0.00313 to 6.40 and 0.0025 to 0.80 ng per tube, respectively. Sensitivities of the assays, defined as the dose at binding (%> = CB, - 2SDB,,) x 100% / CB_ (CB,,: mean cpm of maximal binding, SDB,,: standard deviation of cpm of maximal binding tubes), were 6.25 and 1.56 pg per tube for progesterone and estradiol, respectively. To remove any effects of interassay variability on treatment differences, all samples were assayed in a single assay with intraassay CVs of 5.31 and 6.48% for progesterone and estradiol-17R, respectively.
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Theriogenology
Statistical Analysis Data were analyzed using ANOVA for a randomized, complete block design (replicated trials were blocks; Model Y = block + treatment + error, 18). Percentage data were subjected to arcsine transformation before analysis. Statistical analysis was carried out using the general linear model procedures of the Statistical Analysis System (Version 6.07, SAS Institute, Cary, NC). Multiple comparisons were made by the PDIFF (LSD) function of SAS. Orthogonal contrast analysis was also used to analyze main effects in Experiment 1 (FSH-stimulated vs FSH-LHPRL-stimulated conditioned media; further addition of FSH-LH-PRL during oocyte maturation vs no FSH-LH-PRL addition; 18). RESULTS Experiment
1
Table 2 shows the effect of follicular conditioned media on oocyte maturation. The proportion of oocytes developing beyond the second metaphase (>MII) and the penetrability of MIl oocytes were not affected by supplementation treatment. Male pronuclear development in penetrated oocytes, however, was significantly affected by treatment (Table 2). Multiple comparisons showed that the high rate of male pronuclear formation in penetrated oocytes matured in FSH-stimulated follicular conditioned medium supplemented with the FSH-LH-PRL mixture during maturation culture (Treatment 2) was similar to that in oocytes matured in fresh maturation medium co-cultured with one follicular shell (Treatment 6, P=O.82), but was significantly higher than in oocytes matured in fresh maturation medium without co-culture with a follicular shell (Treatment 5; P=O.O5). Oocytes matured in FSH-LH-PRL-stimulated follicular conditioned medium (Treatments 3 and 4) had lowest male pronuclear formation rates. Orthogonal contrasts also showed that oocytes matured in FSH-stimulated follicular conditioned medium (Treatments 1 and 2) had a significantly higher male pronuclear formation rate than those oocytes matured in FSH-LI-I-PRL-stimulated follicular conditioned medium (Treatments 3 and 4, P=O.O.OOl). Furthermore, addition of the FSH-LH-PRL mixture during the oocyte maturation period may increase the male pronuclear formation rate (comparison of Treatments 2 and 4 with Treatments 1 and 3; P=O.O6). With respect to the steroid content of conditioned media neither estradiol concentrations [13.76*2.98 (n=4) and 18.75f2.47 (n=4) q/ml, respectively] nor progesterone concentrations [537.46&136.3 (n=4) and 864.6f346.0 (n=4) @ml, respectively] in FSH- and FSH-LH-PRLstimulated follicular conditioned media were significantly different (lXt.19). Experiment
2
Table 3 shows that the treatments did not alter nuclear maturation rates (Pa.05) or penetrability (PM.05) of in vitro matured oocytes. However, male pronuclear formation rate was higher in oocytes matured in both conditioned media than in those matured in fresh culture medium (PcO.05). Male pronuclear formation rates were similar in both the first 24-h and the second 24-h groups, and were similar to those matured in FSH-stimulated follicular conditioned medium supplemented with FSH-LH-PRL during oocyte maturation in Experiment 1.
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Theriogenology
Table 2. Effect of follicular shell conditioned medium on oocyte nuclear maturation (WI), sperm penetration and male pronuclear development (MPN)
Treatments
Culture medium
1
FSH-stimulated
FSH-LH-PRL addition Number of oocytes (mean%~SEI@ during Examined oocyte >MII penetrated with MPN maturation -
63 (93.Z.2)d
+
2
3
FSH-LH-PRLstimulated
+
4
5
6
-
Fresh medium
Fresh medium + IFS
+
+
62
68
(%.E3A)d
58 (%.c+4.O)d
(7LZ9.7)d
62 (92.3*5.7)6
60 (97.5f2.qd
(28.iz7.7)’
(94.Z2.5)d
(93.::6.7,”
(40.:8.6)c1
64 (98.8f1.2)’
(95Z4.5)d
(4Lz8.4)d
(92.32.O)d
59 (98.5f1.5)d
(71.42615.O)d
62
65
64
’ The experiment was replicated for five times. b Mean percentage of >MII oocytes: mean of the percentage of the total number of oocytes examined that developed beyond MII; Mean percentage of penetrated oocytes: mean of the percentage of all >MII oocytes penetrated by spermatozoa; and Mean percentage of IWN oocytes: mean of the percentage of all penetrated MI1 oocytes with MPN. ’ One foIlicuIar shell was co-cultured with oocytes. d-fMeans within a column without common superscripts were significantly different (P < 0.05).
The progesterone concentration of 179.05i21.3 q/ml (n=6) in the first 24-h conditioned medium was significantly lower than that of 307.51f50.22 rig/ml (n=7) in the second 24-h conditioned medium (P=O.O46). Conversely, the estradiol concentration of 15.76f1.66 ng/ml (n=7) in the first 24-h conditioned medium was higher than that of 10.48f1.78 ng/ml (n=6) in the second 24-h conditioned medium (P=O.O43). DISCUSSION Follicular secretions obtained from culture of follicular shells under the stimulation of FSH, LH and PRL have been reported to enhance cytoplasmic maturation in the porcine
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Theriogenology
Table 3. Effects of follicular secretions produced during the different culture periods under the stimulation of FSH on oocyte maturation @III), sperm penetration and male pronuclear development (MPN).
Treatment groups
No. replicates
No. oocytes examined
No. oocytes >MII (mean%*seY
No. oocytes penetrated (mean%*seY
No. oocytes With MPN (mean%fse)
1) First 24-h*
8
75
72 (95.2f2.6)”
56 (79.9f8.5)’
36 (61.51t5.8)”
2) Second 24-hd
8
86
73 (85.3f3.9)
(83. p:.,)
40 (66.0f9.0)
(95.c3.8)
(88.87z4.8)
(37.Z9.3)’
3) Fresh Medium
8
84
’ Mean of the percentage of the total number of oocytes developed beyond the MI1 stage (>MII). b Mean of the percentage of all >MII oocytes penetrated by spermatozoa. ’ Mean of the percentage of all penetrated MII oocytes with MPN. * First 24-h: conditioned medium collected after 24 h of culture of 10 follicular shells in 10 ml of oocyte maturation medium supplemented with 2.5 pgknl FSH, Second 24-h: after collection of the first 24-h conditioned medium, the follicular shells were cultured for a second 24 h in 10 ml of fresh oocyte maturation medium containing 2.5 &ml FSH and then conditioned medium was harvested. e-l Means within a column without common superscripts were significantly different (P < 0.05).
oocyte when compared with the control medium (10-l 1). Our results, however, did not confirm this effect. The present results show that follicular secretions stimulated by FSH alone supported cytoplasmic maturation more effectively than follicular secretions stimulated by a combination of FSH, LH and PRL, and which was comparable to the results obtained from co-culture with follicular shells. If anything, follicular secretions stimulated by a combination of FSH, LH and PRL (similar to the treatment of Mattioli et al. 10-l 1) exerted an inhibitory effect on cytoplasmic maturation when compared with fresh culture medium. The precise reasons for this are not clear. However, several differences exist between the 2 culture systems. In our study the ocytes were fixed 10 to 11 h after insemination, while in the above studies (10-l 1) they were fixed at 14 h. Cumulus-enclosed oocytes associated with a piece of mural granulosa were used as a source of immature oocytes in our culture, while cumulus-enclosed oocytes were used by Mattioli et al. (10-l 1). Follicular tissue and oocytes used in our cultures were obtained from prepubertal gilts, as compared with cyclic gilts. Finally, follicular conditioned media in our study were produced by culturing follicular shells alone; Mattioli et al. (10-11) produced conditioned media in the presence of oocytes, and factors resulting from interactions between follicular cells and oocytes were therefore probably involved.
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Theriogenology
The reason for FSH-stimulated follicular secretions promoting cytoplasmic maturation is not known. Physiologically, FSH effects are dominant before the time of the LH surge. Granulosa and theta cells stimulated by FSH at this stage are able to convert androgens of thecal origin to estrogen, resulting in an estrogen-dominated environment in the follicle. After the LH surge, steroid production is substantially altered. Production of estrogen declines and production of progesterone increases (1, 8, 9). Estradiol and progesterone production by follicular shells stimulated by FSH alone or by a combination of FSH, LH and PRL in our present study were not significantly different (P > 0.05), although there was a trend for follicular shells stimulated by FSH alone to be. less steroidogenic than when stimulated by a mixture of FSH, LH and PRL (estradiol: 13.76k2.98 to 18.75k2.47; progesterone: 537.46f136.3 to 864.6f346.0 ng/ml). It seems that follicular cells stimulated by FSH alone also underwent luteinization during culture, which is not comparable to the in vivo situation. This is probably caused by LH contamination (~1%) in FSH (USDA-pFSH-B-1, AFP-5600). The overall change in the progesterone to estradiol ratio in both conditioned media were similar to those found in follicular fluid just before ovulation (100 to 108 h/30 to 36 h post eCG/hCG; 1). Mattioli et al. (11) showed that steroid hormones (progesterone in particular) are mainly responsible for the improvement in cytoplasmic maturation of oocytes matured in conditioned medium. Our results do not imply this. Rather, they further support earlier suggestions that nonsteroidal follicular factors are involved in the regulation of oocyte cytoplasmic maturation (4). Further supplementation of a mixture of FSH, LH and PRL with both types of conditioned media during oocyte culture marginally improved the degree of cytoplasmic maturation of oocytes (P=O.O6, Table 2). It has been reported by Mattioli et al. (12) that oocytes matured in medium containing FSH only had a low male pronuclear formation rate (440/o), while those matured in medium containing LH had a high male pronuclear formation rate (72%). Thus the effect of further supplementation of gonadotropins during oocyte maturation, in conditioned medium stimulated by FSH alone, may be explained by an LH effect on cytoplasmic maturation. However, this would not explain an effect in oocytes matured in conditioned medium stimulated by a mixture of FSH, LH and PRL. One possibility is that the hormones had been used up by follicular cells during the follicle conditioning period. Conditioned medium produced in the first or second 24-h period of culture in the presence of FSH had a similar effect on oocyte nuclear and cytoplasmic maturation, suggesting that the concentrations of stimulating factors in these 2 conditioned media am similar, although the degree of luteinization of follicular cells was lower in the first 24-h period than in the second 24-h period of culture. This suggests that steroidal factors may not be the most critical regulators of cytoplasmic maturation. In summary, the results of our study suggest that FSH-stimulated highly suitable for supporting porcine oocyte maturation in vitro.
follicular secretions are
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Theriogenology REFERENCES
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