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Morphology of porcine cumulus-oocyte-complexes depends on the stage of preovulatory maturation
,v
ELSEVIER
MORPHOLOGY OF PORCINE CUMULUS-OOCYTE-COMPLEXES DEPENDS ON THE STAGE OF PREOVULATORY MATURATION H. Torner, la K.-P. B~ssow, 1 H. Aim, 1 J. Rhtky2 and W, KanitzI Department of Reproductive Biology, Research Institute for the Biology of Farm Animals 18196 Dummerstorf, Germany 2 . • • Department of Reproductive Biology, Research Institute for Animal Breeding and Nutrition 2053 Herceghalom, Hungary Received for publication: July 9, 1997 Accepted: March 23, 1998 ABSTRACT The aim of this investigation was to determine the relationship between the morphology of the cumulus-oocyte-complexes (COCs) and the meiotic configuration of oocytes as an LH peak mimicked by hCG. Estrus was synchronized in a total of 29 crossbred Landrace gilts by feeding Regumate ® for 15 d and administering 1000 IU PMSG. The LH peak was simulated by treatment with 500 IU hCG at 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopically healthy follicles with a diameter of more than 5 mm were punctured. Altogether, 410 follicles from 57 ovaries were punctured and 251 COCs were aspirated. Oocyte recovery rate increased from 48.5 % (P < 0.01) of the early, not yet preovulatory follicles (2 h before hCG) to 80.8 % of late preovulatory follicles (34 h after hCG). Cumulus morphology in COCs recovered 2 h before and 10 h after hCG was heterogeneous, with most (72.9 to 57.4 %; P<0.01) showing a compact or slightly expanded cumulus. Starting at about 22 h after hCG, COC morphology changed dramatically (86.7 % of COCs with expanded cumulus; P<0.01), and 34 h after hCG, 98.3 % of the COCs had only an expanded cumulus. The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2 %, respectively, n=238 oocytes; P<0.01) as the interval post hCG increased (-2, 10, 22, 34 h, respectively). Meiotic configuration was related to COC morphology: compact COCs - 88.9 % diplotene, expanded COCs - 53.8 % metaphase II (M-II), and denuded oocytes - 69.2 % degenerated chromatin. These results indicate that there is a relationship between oocyte recovery rate, COC morphology, and meiotic configuration and preovulatory follicle maturation after the application of hCG. © 1998 by Elsevier Science Inc.
Key words: swine, oocytes, cumulus cell expansion, nuclear maturation. a,Correspondence and reprint requests.
Theriogenology 50:39-48, 1998 © 1998 by Elsevier Science Inc.
0093-691X/98/$19.00 PII S0093-691X(98)00111-3
Theriogenology
40
INTRODUCTION Basic research on oocyte and follicular development and the application of in vitro technology for in vitro maturation and fertilization (IVM/IVF) in pigs are enhanced by techniques for oocyte recovery that are efficient and repeatable. Furthermore, it is necessary to evaluate oocyte maturation in vivo during preovulatory follicular development. There are few studies describing the relationship between oocyte nuclear maturation and cumulus morphology during final follicular maturation in vivo (1, 7, 11, 20). Usually, only one part of the cumulus-oocyte-complex was investigated: qualitative and quantitative structural changes in ooplasm (4) or cumulus cells (18), or changes in follicular endocrinology (25). Little information is available about the stage of meiotic maturation of intrafollicular oocytes recovered at various times during preovulatory development of follicles of gilts treated with PMSG/hCG (1). Precise timing of sequential changes in chromosome configuration during the first meiotic division of pig oocytes was carried out mostly after in vitro maturation (14). Recent research defined some of the controlling mechanisms of oocyte maturation in vitro at the molecular level (8, 9, 13, 15). Nevertheless, further studies of in vivo oocyte maturation in the pig are needed. In the last decade, new diagnostic methods were introduced in studies concerning animal physiology in pigs. One of these methods is laparoscopy, which allows for the recovery of oocytes with less surgical stress. To recover COC at different stages of preovulatory follicular maturation, a laparoscopic technique for repeated follicular puncture and oocyte aspiration in swine was developed (3). The aim of the present study was to determine the influence of the stage of preovulatory maturation in relation to the hCG-simulated LH peak on the recovery rate and morphology of COCs and the meiotic configuration of oocytes. MATERIALS AND METHODS Oocyte Collection A total of 29 crossbred Landrace gilts aged 8.5 months with a body weight of 120 to 125 kg were used. Gilts were synchronized by feeding Regumate ® (20 mg altrenogest/animal daily, Serum-Werk Bemburg, Germany) for 15 d. Follicular growth was stimulated by administering 1,000 IU, im PMSG (Pregmagon ®, Dessau, Germany) 24 h after the last Regumate ® feeding (0800 h). The LH peak was simulated by treatment with 500 IU hCG 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopicaily healthy follicles, well vascularized and translucent and with a diameter of > 5 mm were punctured. Follicular aspiration was carried out as described by Briissow and Rhtky (3) via a twoway cannula (40 mm in length, 16-gauge) and an electric aspiration pump (model 3014, Labotect, Gtttingen, Germany) with an initial vacuum of 100 mm Hg corresponding to a volume of 17 mL/min. The tip of the aspiration cannula was inserted into a follicle, the follicular contents were aspirated and the follicle was refilled and aspirated twice with heparinized PBS.
Theriogenology
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COC Evaluation Follicular fluids from different follicles of one ovary were pooled, and the morphology of 251 freshly recovered COCs was determined using an inverted microscope at x 60 magnification. The COCs were classified as compact (Figure 1), slightly-expanded (Figure 2), expanded (Figure 3), with only a corona radiata (Figure 4), or denuded oocytes (Figure 5). After classifying, the COCs were immediately prepared for evaluation of nuclear configuration. Removal of cumulus cells was accomplished in PBS containing 100 IU/mL hyaluronidase (Hylase, Impfstoffwerk Dessau, Germany) followed repeated pipetting with a fine-bore glass pipette. The oocytes were then mounted on slides and fixed for > 24 h in a mixture of acetic acid/alcohol/chloroform (3:6:1) before staining with 2 % orcein in 60 % acetic acid. The nuclear configuration of 238 oocytes was examined using phase-contrast optics at x 250 to x 630 magnification. Based on their nuclear status the oocytes were classified as 1) immature - germinal vesicle (GV), with diplotene chromatin; 2) meiosis resumed - GV breakdown, diakinesis, M-I to A-I; 3) mature - T-I and M-II; or 4) degenerated - pycnotic chromatin at various meiotic stages. Data were analyzed by Chi-square, and P < 0.05 was considered to be statistically significant.
Figure 1. Pig oocyte with compact cumulus; the oocyte is enclosed by a dark, thickness cumulus investment. (x 130)
42
Thedogeno~gy
Figure 2. Pig oocyte with slightly expanded cumulus; the cumulus investment is starting for expansion. (x 130)
Figure 3. Pig oocyte with expanded cumulus, partially dissociated. (x 130)
Thefiogeno~gy
Figure 4. Pig oocyte only with corona radiata cells. (x 130)
Figure 5. Pig oocyte without cumulus cells. (x 130)
43
Theriogenology
44
RESULTS COC Recovery Rate and Morphology According to Time after hCG The results of follicular puncture and COC recovery at different times before and after the injection ofhCG are presented in Table 1. Table 1. Follicular puncture and oocyte recovery at different times before and after the injection of hCG (n=410 follicles) Time after hCG Number of punctured Number of aspirated Recovery rate injection follicles oocytes (hours) (n) (n) (%) -2
175
85
48.5a
10
70
47
67.1 c
22
92
60
65.2e
34
73
59
80.8b
a:b;a:c P<0.01; e:b P<0.05. Altogether 410 follicles from 57 ovaries were punctured and 251 COCs (61.2 %) were" recovered. The oocyte recovery rate increased (P<0.01) from 48.5 % of early, not yet preovulatory follicles (at 2 h before hCG) to 80.8 % of late preovulatory follicles (at 34 h after hCG; Table 1). Most (72.9 %) COCs aspirated from early, not yet preovulatory follicles (2 h before hCG) were compact or slightly expanded (Table 2), and after hCG, the percentage of compact COCs decreased while the percentage of expanded COCs increased (P<0.01) from 25.5 % (10 h post hCG) to 98.3 % in late preovulatory follicles (34 h post hCG). Although only macroscopicaUy healthy follicles were aspirated, 10.6 % of the COCs were denuded. Table 2. COC morphology at different times before and after hCG injection (n=251 oocytes) Time after Number of COC morphology hCG COC injection Compact Slightly Corona Expanded Denuded expanded radiata (%) (%) (%) (%) (%) (hours) (n) -2
85
42.3
30.6a
18.8
0
8.2
10
47
25.5
31.9a
6.4
25.5c
10.6
22
60
0
5.0b
3.3
86.7d'e
5.0
34
59
0
0
1.7
98.3d'f
0
a:b;e:d P<0.01; e:fp<0.05.
Theriogenology
45
Meiotic Configuration as Related to the Morphology of COCs and Time after hCG The investigation of meiotic configuration as related to COC morphology (Table 3) showed that most of the oocytes from compact COCs were immature (88.9 %). The proportion of oocytes resuming meiosis was highest in oocytes with corona radiata (72.2 %). Also, about half of the oocytes with slightly expanded COC (48.4 %), and the denuded oocytes (53.8 %) had resumed meiosis. The proportion of oocytes with degenerated chromatin was independent of cumulus investment, and varied between 8.9 and 18.7 % compared with 69.2 % in denuded oocytes. Table 3. Meiotic configuration depending on COC morphology (n=236 oocytes) Chromatin configuration (%) COC morphology Number of COC immature resumption of mature (n) (GV) meiosis (GVBD-A I) (T I/M II) Compact
45
88.9a
11.1a
0
Slight expanded
43
32.6b
48.8b
18.6a
Expanded
117
12.0c
34.2b
53.8b
Corona radiata
18
16.7c
72.2b
11.1a
Denuded
13
15.4c
53.8b
30.8a'b
a:b:cP<0.01, 3x5-frequency table. The meiotic configuration in oocytes was evaluated according to their follicular maturation at different times related to the hCG application (Table 4). The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2 %, respectively, n=238 oocytes; P<0.01) in accordance with the level of preovulatory maturation (-2, 10, 22 and 34 h after hCG injection). Most of the oocytes were in immature stages of meiosis at the beginning of preovulatory maturation, i.e., 2 h before to 10 h after hCG. Similar percentages of oocytes resuming meiosis were found at each interval post hCG. Table 4. Meiotic configuration in follicular oocytes as related to post hCG interval (n=238) Chromatin configuration (%) Time after hCG Number of COC immature resumption of mature injection (hours) (n) (GV) meiosis (GVBD-A I) (T I/M II) -2
81
44.4a
44.4
11.2a
10
42
66.6b
26.2
7.1 a
22
58
17.2c
41.4
41.4 b
34
57
0
29.8
70.2c
a:b:e P
Theriogenology
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were mature 10 h after hCG, but 46% and 69.7% (P<0.01) were mature at 22 h and 34 h after hCG, respectively, indicating that cumulus expansion precedes nuclear maturation. DISCUSSION Our study showed that endoscopic follicular aspiration resulted in the collection of a high number of oocytes with an intact cumulus investment (237/251). The overall recovery rate was 4.4 oocytes per ovary. The aspiration volume of 17 mL water/min is optimal for oocyte recovery and COC quality in swine (4). The number of aspirated oocytes and the collection rates (61.2 %) are comparable to the results in cattle. Pieterse et al. (16), Kruip et al. (10), Spitschak et al. (21) and Becker et al. (2) reported recovery rates of 55 %, 47.5 to 56.9 %, 38.5 to 43.5 %, and 44 %, respectively, by means of ultrasound-guided oocyte aspiration in cattle. Slightly higher rates (79 % and 51 to 68 %) were achieved (6, 17) by aspirating oocytes endoscopically. The oocyte recovery rate increased from 48.5 % from early preovulatory follicles (2 h before hCG) to 80.8 % of late preovutatory follicles (34 h after hCG), indicating that in the early preovulatory follicle the compact COC is still tightly attached to the granulosa cells. Granulosa-cumulus uncoupling is evident only after the preovulatory LH surge (23). The cumulus morphology of the COC population recovered 2 h before and 10 h after hCG was heterogeneous, with most of the COCs (72.9 and 57.5 %) showing a compact or slightly expanded cumulus. Similar results were reported by Techakumphu et al. (22), who recovered 6 1 % of the oocytes with compact or slightly expanded cumulus. Cumulus expansion was first observed 10 h after hCG. These results are similar to those of Meinecke et al. (11), who found that the first signals of in vivo preovulatory transformation of the cumulus oophorus are visible 8 h after hCG. We found that cumulus morphology had changed dramatically by 22 h after hCG, and by 34 h after hCG nearly all of the COCs expressed only expanded cumuli. The morphological changes in the pig oocyte during maturation may be broadly divided into 2 phases; a phase up to about 20 h after hCG injection and the subsequent period up to ovulation (5). Our results on the change in the cumulus morphology 22 h after hCG are similar to those reported by Cran (5). During in vivo maturation, intercellular coupling between the oocyte and expanded cumulus persisted up to 32 h after hCG. However, the functional coupling between cumulus cells and the oocyte in vitro was maintained for only 16 h (12). This was accompanied by alterations in the protein synthesis pattern in the cumulus oophorus (19). Most oocytes at each stage of COC morphology had resumed meiosis, most oocytes with compact cumulus were in GV-stage and most oocytes with expanded cumuli were in telophase I or metaphase II, indicating that maturation is related directly to cumulus cell expansion. Nevertheless, it was not possible to categorize the stage of maturation according to
Theriogenology
47
COC morphology alone. Different stages of nuclear maturation were observed in oocytes with a slightly expanded cumulus, in oocytes with corona radiata and in denuded oocytes. In our investigation of the relationship between follicular maturation after hCG and oocyte meiotic status in swine it was confirmed that follicular maturation and oocyte maturation were highly correlated (25). Porcine oocytes with expanded cumulus showed different degrees of nuclear maturation according to the time after hCG, indicating that cumulus expansion precedes nuclear maturation. Similar results were obtained by Ainsworth et al. (1). The percentage of meiotically mature oocytes increased according to the degree of preovulatory maturation from 10 to 34 h after hCG. In comparison with our results for in vivo maturation of porcine oocytes, in vitro maturation was delayed. Oocytes reached the M-II phase 34.4 to 48 h after IVM (14). In cyclic gilts, it was observed that maturation of oocytes during normal estrous cycles started about 18 h and progressed to M-II about 36 h after the onset of estrus (20). In our study we found the oocytes in the metaphase II stage between 22 and 34 h. Xie et al. (24) obtained similar results after in vivo maturation. By 24 h after the administration of hCG, meiosis proceeded to M-I and A-I/T-I, and continued to M-II by 27 to 30 h. In conclusion, our results indicate a relationship between oocyte recovery rate, cumulus morphology and oocyte meiotic maturation and hCG-induced preovulatory follicle maturation. REFERENCES 1. Ainsworth L, Tsang BK, Downey BR, Marcus GJ, Armstrong 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. Becker F, Kanitz W, NOrnberg G, Kurt J, Spitschak M. Comparison of repeated transvaginal ovum pick up in heifers by ultrasonographic and endoscopic instruments. Theriogenology 1996; 46:999-1007. 3. Briissow KP, Rhtky J. Repeated laparoscopical follicular puncture and oocyte aspiration in swine. Reprod Dom Anita 1994;29:494-502. 4. Briissow KP, Torner H, R~ttky J, Hunter MG, Ntirnberg G. Ovum pick up in swine: The influence of aspiration vacuum on the morphology of cumulus oocyte complexes and on oocyte recovery from preovulatory follicles. Acta Vet Hung 1997;45. 5. Cran DG. Qualitative and quantitative structural changes during pig oocyte maturation. J Reprod Fertil 1985;74:237-245. 6. Fayrer-Hosken R.A, Candle AB. The laparoscope in follicular oocyte collection and gamete intrafallopian transfer and fertilization (GIFT). Theriogenology 1991;36:709725.
48
Theriogenology
7. Flechon JE, Motlik J, Hunter RH, Fechon B, Pivko J, Fulka J. Cumulus oophorus mucification during resumption of meiosis in th pig. A scanning electron microscope study. Reprod Nutr Dev 1986;26:989-98. 8. Homa ST. Calcium and meiotic maturation of the mammalian oocyte, Mol Reprod Dev 1995;40:122-134. 9. Inoue M, Naito K, Aoki F, Yoyoda Y, Sato E. Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes. Zygote 1995;3:265-271. 10. Kruip TAM, Boni R, Roelofsen MWM, Wuth YA, Pieterse MC. Application of OPU for embryo production and breeding in cattle. Theriogenology 1993;39:251. 11. Meinecke B, Meinecke-Tillmann S, Gips H. Untersuchungen zur biomorphologischen Differenzierung des Cumulus-Oocyten-Komplexes des Schweines wahrend der pr~iovulatorischen Follikelreifung. Zbl Vet Med A 1984;31:370-385. 12. Motlik J, Fulka J, F16chon JE. Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro. J Reprod Fertil 1986;76:31-37. 13. Naito K, Toyoda Y. Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. J Reprod Fertil 1991 ;93:467-473. 14. Ocampo MB, Ocampo LT, Kanagawa H. Timing of sequential changes in chromosome configurations during the 1st meiotic division of pig oocytes cultured in vitro. Jpn J Vet Res 1990;38:127-137. 15. Petr J, Tepl~i O, Grocholov~t R, Jilek F. Inhibition of meiotic maturation in growing pig oocytes by factor(s) from cumulus cells. Reprod Nutr Dev 1994;34:149-156. 16. Pieterse MC, Vos PLAM, Kruip TAM, Willemse AH, Taverne MAM. Characteristics of bovine estrous cycles during repeated transvaginal, ultrasound-guided puncturing of follicles for ovum pick-up. Theriogenology 1991 ;35:401-413. 17. Reichenbach HD, Wiebke NH, Besenfelder UH, M/kdl J, Brem G. Transvaginal laparoscopic guided aspiration of bovine follicular oocytes: Preliminary results. Theriogenology 1993;39:295. 18. Schnurrbusch U, Elze K. Ober das Verhalten der Cumuluszellen in reifenden Follikeln des Schweines w~ihrend des unbeeinflufSten Zyklus. Wien Tier~ztl Mschr 1989;76:69-79. 19. Schroeter D, Meinecke B. Comparative analysis of the polypeptide pattern of cumulus ceils during maturation of porcine cumulus oocyte complexes in vivo and in vitro. Reprod Nutr Dev 1995;35:85-94. 20. Spalding JF, Berry RO, Moffit JG. The maturation process of the ovum of swine during normal and induced ovulations. J Anim Sci 1955;14:609-620. 21. Spitschak M, Becker F, Kanitz W. UltraschaUgesttitzte Untersuchungen zum Follikelwachstum und Oozytenreifung beim Rind. Reprod Dom Anim 1993;28:167. 22, Techakumphu M, Srianan W, Singlor J, Tantasuparak W. Improvement of in vitro fertilization in pig by using diluted sperm. Theriogenology 1994;41:314. 23, Thibault C, SzSllOsi D, G6rard M. Mammalian oocyte maturation. Reprod Nutr Dev 1987;27:865-896. 24. Xie S, Broermann DM, Nephew KP, Bishop MD, Pope WF. Relationship between oocyte maturation and fertilization on zygotic diversity in swine. J Anim Sci 1990;68:20272033. 25. Xie S, Broermann DM, Nephew KP, Ottobre JS, Day ML, Pope WF. Changes in follicular endocrinology during final maturation of porcine oocytes. Dora Anim Endocrinol 1990;7:75-82.
ELSEVIER
MORPHOLOGY OF PORCINE CUMULUS-OOCYTE-COMPLEXES DEPENDS ON THE STAGE OF PREOVULATORY MATURATION H. Torner, la K.-P. B~ssow, 1 H. Aim, 1 J. Rhtky2 and W, KanitzI Department of Reproductive Biology, Research Institute for the Biology of Farm Animals 18196 Dummerstorf, Germany 2 . • • Department of Reproductive Biology, Research Institute for Animal Breeding and Nutrition 2053 Herceghalom, Hungary Received for publication: July 9, 1997 Accepted: March 23, 1998 ABSTRACT The aim of this investigation was to determine the relationship between the morphology of the cumulus-oocyte-complexes (COCs) and the meiotic configuration of oocytes as an LH peak mimicked by hCG. Estrus was synchronized in a total of 29 crossbred Landrace gilts by feeding Regumate ® for 15 d and administering 1000 IU PMSG. The LH peak was simulated by treatment with 500 IU hCG at 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopically healthy follicles with a diameter of more than 5 mm were punctured. Altogether, 410 follicles from 57 ovaries were punctured and 251 COCs were aspirated. Oocyte recovery rate increased from 48.5 % (P < 0.01) of the early, not yet preovulatory follicles (2 h before hCG) to 80.8 % of late preovulatory follicles (34 h after hCG). Cumulus morphology in COCs recovered 2 h before and 10 h after hCG was heterogeneous, with most (72.9 to 57.4 %; P<0.01) showing a compact or slightly expanded cumulus. Starting at about 22 h after hCG, COC morphology changed dramatically (86.7 % of COCs with expanded cumulus; P<0.01), and 34 h after hCG, 98.3 % of the COCs had only an expanded cumulus. The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2 %, respectively, n=238 oocytes; P<0.01) as the interval post hCG increased (-2, 10, 22, 34 h, respectively). Meiotic configuration was related to COC morphology: compact COCs - 88.9 % diplotene, expanded COCs - 53.8 % metaphase II (M-II), and denuded oocytes - 69.2 % degenerated chromatin. These results indicate that there is a relationship between oocyte recovery rate, COC morphology, and meiotic configuration and preovulatory follicle maturation after the application of hCG. © 1998 by Elsevier Science Inc.
Key words: swine, oocytes, cumulus cell expansion, nuclear maturation. a,Correspondence and reprint requests.
Theriogenology 50:39-48, 1998 © 1998 by Elsevier Science Inc.
0093-691X/98/$19.00 PII S0093-691X(98)00111-3
Theriogenology
40
INTRODUCTION Basic research on oocyte and follicular development and the application of in vitro technology for in vitro maturation and fertilization (IVM/IVF) in pigs are enhanced by techniques for oocyte recovery that are efficient and repeatable. Furthermore, it is necessary to evaluate oocyte maturation in vivo during preovulatory follicular development. There are few studies describing the relationship between oocyte nuclear maturation and cumulus morphology during final follicular maturation in vivo (1, 7, 11, 20). Usually, only one part of the cumulus-oocyte-complex was investigated: qualitative and quantitative structural changes in ooplasm (4) or cumulus cells (18), or changes in follicular endocrinology (25). Little information is available about the stage of meiotic maturation of intrafollicular oocytes recovered at various times during preovulatory development of follicles of gilts treated with PMSG/hCG (1). Precise timing of sequential changes in chromosome configuration during the first meiotic division of pig oocytes was carried out mostly after in vitro maturation (14). Recent research defined some of the controlling mechanisms of oocyte maturation in vitro at the molecular level (8, 9, 13, 15). Nevertheless, further studies of in vivo oocyte maturation in the pig are needed. In the last decade, new diagnostic methods were introduced in studies concerning animal physiology in pigs. One of these methods is laparoscopy, which allows for the recovery of oocytes with less surgical stress. To recover COC at different stages of preovulatory follicular maturation, a laparoscopic technique for repeated follicular puncture and oocyte aspiration in swine was developed (3). The aim of the present study was to determine the influence of the stage of preovulatory maturation in relation to the hCG-simulated LH peak on the recovery rate and morphology of COCs and the meiotic configuration of oocytes. MATERIALS AND METHODS Oocyte Collection A total of 29 crossbred Landrace gilts aged 8.5 months with a body weight of 120 to 125 kg were used. Gilts were synchronized by feeding Regumate ® (20 mg altrenogest/animal daily, Serum-Werk Bemburg, Germany) for 15 d. Follicular growth was stimulated by administering 1,000 IU, im PMSG (Pregmagon ®, Dessau, Germany) 24 h after the last Regumate ® feeding (0800 h). The LH peak was simulated by treatment with 500 IU hCG 80 h after PMSG. Endoscopic oocyte recovery was carried out 2 h before and 10, 22 and 34 h after hCG. Only macroscopicaily healthy follicles, well vascularized and translucent and with a diameter of > 5 mm were punctured. Follicular aspiration was carried out as described by Briissow and Rhtky (3) via a twoway cannula (40 mm in length, 16-gauge) and an electric aspiration pump (model 3014, Labotect, Gtttingen, Germany) with an initial vacuum of 100 mm Hg corresponding to a volume of 17 mL/min. The tip of the aspiration cannula was inserted into a follicle, the follicular contents were aspirated and the follicle was refilled and aspirated twice with heparinized PBS.
Theriogenology
41
COC Evaluation Follicular fluids from different follicles of one ovary were pooled, and the morphology of 251 freshly recovered COCs was determined using an inverted microscope at x 60 magnification. The COCs were classified as compact (Figure 1), slightly-expanded (Figure 2), expanded (Figure 3), with only a corona radiata (Figure 4), or denuded oocytes (Figure 5). After classifying, the COCs were immediately prepared for evaluation of nuclear configuration. Removal of cumulus cells was accomplished in PBS containing 100 IU/mL hyaluronidase (Hylase, Impfstoffwerk Dessau, Germany) followed repeated pipetting with a fine-bore glass pipette. The oocytes were then mounted on slides and fixed for > 24 h in a mixture of acetic acid/alcohol/chloroform (3:6:1) before staining with 2 % orcein in 60 % acetic acid. The nuclear configuration of 238 oocytes was examined using phase-contrast optics at x 250 to x 630 magnification. Based on their nuclear status the oocytes were classified as 1) immature - germinal vesicle (GV), with diplotene chromatin; 2) meiosis resumed - GV breakdown, diakinesis, M-I to A-I; 3) mature - T-I and M-II; or 4) degenerated - pycnotic chromatin at various meiotic stages. Data were analyzed by Chi-square, and P < 0.05 was considered to be statistically significant.
Figure 1. Pig oocyte with compact cumulus; the oocyte is enclosed by a dark, thickness cumulus investment. (x 130)
42
Thedogeno~gy
Figure 2. Pig oocyte with slightly expanded cumulus; the cumulus investment is starting for expansion. (x 130)
Figure 3. Pig oocyte with expanded cumulus, partially dissociated. (x 130)
Thefiogeno~gy
Figure 4. Pig oocyte only with corona radiata cells. (x 130)
Figure 5. Pig oocyte without cumulus cells. (x 130)
43
Theriogenology
44
RESULTS COC Recovery Rate and Morphology According to Time after hCG The results of follicular puncture and COC recovery at different times before and after the injection ofhCG are presented in Table 1. Table 1. Follicular puncture and oocyte recovery at different times before and after the injection of hCG (n=410 follicles) Time after hCG Number of punctured Number of aspirated Recovery rate injection follicles oocytes (hours) (n) (n) (%) -2
175
85
48.5a
10
70
47
67.1 c
22
92
60
65.2e
34
73
59
80.8b
a:b;a:c P<0.01; e:b P<0.05. Altogether 410 follicles from 57 ovaries were punctured and 251 COCs (61.2 %) were" recovered. The oocyte recovery rate increased (P<0.01) from 48.5 % of early, not yet preovulatory follicles (at 2 h before hCG) to 80.8 % of late preovulatory follicles (at 34 h after hCG; Table 1). Most (72.9 %) COCs aspirated from early, not yet preovulatory follicles (2 h before hCG) were compact or slightly expanded (Table 2), and after hCG, the percentage of compact COCs decreased while the percentage of expanded COCs increased (P<0.01) from 25.5 % (10 h post hCG) to 98.3 % in late preovulatory follicles (34 h post hCG). Although only macroscopicaUy healthy follicles were aspirated, 10.6 % of the COCs were denuded. Table 2. COC morphology at different times before and after hCG injection (n=251 oocytes) Time after Number of COC morphology hCG COC injection Compact Slightly Corona Expanded Denuded expanded radiata (%) (%) (%) (%) (%) (hours) (n) -2
85
42.3
30.6a
18.8
0
8.2
10
47
25.5
31.9a
6.4
25.5c
10.6
22
60
0
5.0b
3.3
86.7d'e
5.0
34
59
0
0
1.7
98.3d'f
0
a:b;e:d P<0.01; e:fp<0.05.
Theriogenology
45
Meiotic Configuration as Related to the Morphology of COCs and Time after hCG The investigation of meiotic configuration as related to COC morphology (Table 3) showed that most of the oocytes from compact COCs were immature (88.9 %). The proportion of oocytes resuming meiosis was highest in oocytes with corona radiata (72.2 %). Also, about half of the oocytes with slightly expanded COC (48.4 %), and the denuded oocytes (53.8 %) had resumed meiosis. The proportion of oocytes with degenerated chromatin was independent of cumulus investment, and varied between 8.9 and 18.7 % compared with 69.2 % in denuded oocytes. Table 3. Meiotic configuration depending on COC morphology (n=236 oocytes) Chromatin configuration (%) COC morphology Number of COC immature resumption of mature (n) (GV) meiosis (GVBD-A I) (T I/M II) Compact
45
88.9a
11.1a
0
Slight expanded
43
32.6b
48.8b
18.6a
Expanded
117
12.0c
34.2b
53.8b
Corona radiata
18
16.7c
72.2b
11.1a
Denuded
13
15.4c
53.8b
30.8a'b
a:b:cP<0.01, 3x5-frequency table. The meiotic configuration in oocytes was evaluated according to their follicular maturation at different times related to the hCG application (Table 4). The percentage of oocytes with a mature meiotic configuration increased (11.2; 7.1; 41.4 and 70.2 %, respectively, n=238 oocytes; P<0.01) in accordance with the level of preovulatory maturation (-2, 10, 22 and 34 h after hCG injection). Most of the oocytes were in immature stages of meiosis at the beginning of preovulatory maturation, i.e., 2 h before to 10 h after hCG. Similar percentages of oocytes resuming meiosis were found at each interval post hCG. Table 4. Meiotic configuration in follicular oocytes as related to post hCG interval (n=238) Chromatin configuration (%) Time after hCG Number of COC immature resumption of mature injection (hours) (n) (GV) meiosis (GVBD-A I) (T I/M II) -2
81
44.4a
44.4
11.2a
10
42
66.6b
26.2
7.1 a
22
58
17.2c
41.4
41.4 b
34
57
0
29.8
70.2c
a:b:e P
Theriogenology
46
were mature 10 h after hCG, but 46% and 69.7% (P<0.01) were mature at 22 h and 34 h after hCG, respectively, indicating that cumulus expansion precedes nuclear maturation. DISCUSSION Our study showed that endoscopic follicular aspiration resulted in the collection of a high number of oocytes with an intact cumulus investment (237/251). The overall recovery rate was 4.4 oocytes per ovary. The aspiration volume of 17 mL water/min is optimal for oocyte recovery and COC quality in swine (4). The number of aspirated oocytes and the collection rates (61.2 %) are comparable to the results in cattle. Pieterse et al. (16), Kruip et al. (10), Spitschak et al. (21) and Becker et al. (2) reported recovery rates of 55 %, 47.5 to 56.9 %, 38.5 to 43.5 %, and 44 %, respectively, by means of ultrasound-guided oocyte aspiration in cattle. Slightly higher rates (79 % and 51 to 68 %) were achieved (6, 17) by aspirating oocytes endoscopically. The oocyte recovery rate increased from 48.5 % from early preovulatory follicles (2 h before hCG) to 80.8 % of late preovutatory follicles (34 h after hCG), indicating that in the early preovulatory follicle the compact COC is still tightly attached to the granulosa cells. Granulosa-cumulus uncoupling is evident only after the preovulatory LH surge (23). The cumulus morphology of the COC population recovered 2 h before and 10 h after hCG was heterogeneous, with most of the COCs (72.9 and 57.5 %) showing a compact or slightly expanded cumulus. Similar results were reported by Techakumphu et al. (22), who recovered 6 1 % of the oocytes with compact or slightly expanded cumulus. Cumulus expansion was first observed 10 h after hCG. These results are similar to those of Meinecke et al. (11), who found that the first signals of in vivo preovulatory transformation of the cumulus oophorus are visible 8 h after hCG. We found that cumulus morphology had changed dramatically by 22 h after hCG, and by 34 h after hCG nearly all of the COCs expressed only expanded cumuli. The morphological changes in the pig oocyte during maturation may be broadly divided into 2 phases; a phase up to about 20 h after hCG injection and the subsequent period up to ovulation (5). Our results on the change in the cumulus morphology 22 h after hCG are similar to those reported by Cran (5). During in vivo maturation, intercellular coupling between the oocyte and expanded cumulus persisted up to 32 h after hCG. However, the functional coupling between cumulus cells and the oocyte in vitro was maintained for only 16 h (12). This was accompanied by alterations in the protein synthesis pattern in the cumulus oophorus (19). Most oocytes at each stage of COC morphology had resumed meiosis, most oocytes with compact cumulus were in GV-stage and most oocytes with expanded cumuli were in telophase I or metaphase II, indicating that maturation is related directly to cumulus cell expansion. Nevertheless, it was not possible to categorize the stage of maturation according to
Theriogenology
47
COC morphology alone. Different stages of nuclear maturation were observed in oocytes with a slightly expanded cumulus, in oocytes with corona radiata and in denuded oocytes. In our investigation of the relationship between follicular maturation after hCG and oocyte meiotic status in swine it was confirmed that follicular maturation and oocyte maturation were highly correlated (25). Porcine oocytes with expanded cumulus showed different degrees of nuclear maturation according to the time after hCG, indicating that cumulus expansion precedes nuclear maturation. Similar results were obtained by Ainsworth et al. (1). The percentage of meiotically mature oocytes increased according to the degree of preovulatory maturation from 10 to 34 h after hCG. In comparison with our results for in vivo maturation of porcine oocytes, in vitro maturation was delayed. Oocytes reached the M-II phase 34.4 to 48 h after IVM (14). In cyclic gilts, it was observed that maturation of oocytes during normal estrous cycles started about 18 h and progressed to M-II about 36 h after the onset of estrus (20). In our study we found the oocytes in the metaphase II stage between 22 and 34 h. Xie et al. (24) obtained similar results after in vivo maturation. By 24 h after the administration of hCG, meiosis proceeded to M-I and A-I/T-I, and continued to M-II by 27 to 30 h. In conclusion, our results indicate a relationship between oocyte recovery rate, cumulus morphology and oocyte meiotic maturation and hCG-induced preovulatory follicle maturation. REFERENCES 1. Ainsworth L, Tsang BK, Downey BR, Marcus GJ, Armstrong 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. Becker F, Kanitz W, NOrnberg G, Kurt J, Spitschak M. Comparison of repeated transvaginal ovum pick up in heifers by ultrasonographic and endoscopic instruments. Theriogenology 1996; 46:999-1007. 3. Briissow KP, Rhtky J. Repeated laparoscopical follicular puncture and oocyte aspiration in swine. Reprod Dom Anita 1994;29:494-502. 4. Briissow KP, Torner H, R~ttky J, Hunter MG, Ntirnberg G. Ovum pick up in swine: The influence of aspiration vacuum on the morphology of cumulus oocyte complexes and on oocyte recovery from preovulatory follicles. Acta Vet Hung 1997;45. 5. Cran DG. Qualitative and quantitative structural changes during pig oocyte maturation. J Reprod Fertil 1985;74:237-245. 6. Fayrer-Hosken R.A, Candle AB. The laparoscope in follicular oocyte collection and gamete intrafallopian transfer and fertilization (GIFT). Theriogenology 1991;36:709725.
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7. Flechon JE, Motlik J, Hunter RH, Fechon B, Pivko J, Fulka J. Cumulus oophorus mucification during resumption of meiosis in th pig. A scanning electron microscope study. Reprod Nutr Dev 1986;26:989-98. 8. Homa ST. Calcium and meiotic maturation of the mammalian oocyte, Mol Reprod Dev 1995;40:122-134. 9. Inoue M, Naito K, Aoki F, Yoyoda Y, Sato E. Activation of mitogen-activated protein kinase during meiotic maturation in porcine oocytes. Zygote 1995;3:265-271. 10. Kruip TAM, Boni R, Roelofsen MWM, Wuth YA, Pieterse MC. Application of OPU for embryo production and breeding in cattle. Theriogenology 1993;39:251. 11. Meinecke B, Meinecke-Tillmann S, Gips H. Untersuchungen zur biomorphologischen Differenzierung des Cumulus-Oocyten-Komplexes des Schweines wahrend der pr~iovulatorischen Follikelreifung. Zbl Vet Med A 1984;31:370-385. 12. Motlik J, Fulka J, F16chon JE. Changes in intercellular coupling between pig oocytes and cumulus cells during maturation in vivo and in vitro. J Reprod Fertil 1986;76:31-37. 13. Naito K, Toyoda Y. Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. J Reprod Fertil 1991 ;93:467-473. 14. Ocampo MB, Ocampo LT, Kanagawa H. Timing of sequential changes in chromosome configurations during the 1st meiotic division of pig oocytes cultured in vitro. Jpn J Vet Res 1990;38:127-137. 15. Petr J, Tepl~i O, Grocholov~t R, Jilek F. Inhibition of meiotic maturation in growing pig oocytes by factor(s) from cumulus cells. Reprod Nutr Dev 1994;34:149-156. 16. Pieterse MC, Vos PLAM, Kruip TAM, Willemse AH, Taverne MAM. Characteristics of bovine estrous cycles during repeated transvaginal, ultrasound-guided puncturing of follicles for ovum pick-up. Theriogenology 1991 ;35:401-413. 17. Reichenbach HD, Wiebke NH, Besenfelder UH, M/kdl J, Brem G. Transvaginal laparoscopic guided aspiration of bovine follicular oocytes: Preliminary results. Theriogenology 1993;39:295. 18. Schnurrbusch U, Elze K. Ober das Verhalten der Cumuluszellen in reifenden Follikeln des Schweines w~ihrend des unbeeinflufSten Zyklus. Wien Tier~ztl Mschr 1989;76:69-79. 19. Schroeter D, Meinecke B. Comparative analysis of the polypeptide pattern of cumulus ceils during maturation of porcine cumulus oocyte complexes in vivo and in vitro. Reprod Nutr Dev 1995;35:85-94. 20. Spalding JF, Berry RO, Moffit JG. The maturation process of the ovum of swine during normal and induced ovulations. J Anim Sci 1955;14:609-620. 21. Spitschak M, Becker F, Kanitz W. UltraschaUgesttitzte Untersuchungen zum Follikelwachstum und Oozytenreifung beim Rind. Reprod Dom Anim 1993;28:167. 22, Techakumphu M, Srianan W, Singlor J, Tantasuparak W. Improvement of in vitro fertilization in pig by using diluted sperm. Theriogenology 1994;41:314. 23, Thibault C, SzSllOsi D, G6rard M. Mammalian oocyte maturation. Reprod Nutr Dev 1987;27:865-896. 24. Xie S, Broermann DM, Nephew KP, Bishop MD, Pope WF. Relationship between oocyte maturation and fertilization on zygotic diversity in swine. J Anim Sci 1990;68:20272033. 25. Xie S, Broermann DM, Nephew KP, Ottobre JS, Day ML, Pope WF. Changes in follicular endocrinology during final maturation of porcine oocytes. Dora Anim Endocrinol 1990;7:75-82.