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Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles
Theriogenology 65 (2006) 1422–1434 www.journals.elsevierhealth.com/periodicals/the
Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles H. Alm a,*, L. Ka˛tska-Ksia˛z˙kiewicz b, B. Ryn´ska b, A. Tuchscherer a a
Forschungsinstitut fu¨r die Biologie Landwirtschaftlicher Nutztiere, 18196 Dummerstorf, Rostock, Germany Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, Balice, Krakow, Poland
b
Received 23 March 2005; received in revised form 29 August 2005; accepted 30 August 2005
Abstract The aim of the present study was to examine the growth and survival in culture, and the subsequent meiotic competence, of bovine oocytes recovered from early antral ovarian follicles. Follicles isolated by microdissection of the ovarian slices were sorted into two size groups: (I) 0.2–0.5 mm diameter; and (II) 0.4–0.7 mm diameter. Group I follicles were cultured intact while in Group II, cumulus–oocyte complexes with pieces of parietal granulosa were dissected from the follicles and cultured. Follicles or cumulus–oocyte complexes with parietal granulose were embedded in collagen gel and cultured in TCM 199 supplemented with 3% BSA and 4 mM hypoxanthine for 14 days (Group I) or 7–10 days (Group II). After this, cumulus–oocyte complexes were recovered from the gel. Oocytes that had lost the majority of the cumulus were fixed immediately after recovery. Cumulus–oocyte complexes showing normal morphology were either fixed immediately or were subjected to IVM for an additional 24 h, and then were fixed. At the end of the growth culture, 57.6% of the compact COCs in Group I follicles were preserved in the GV configuration, 16.7% had resumed meiosis, and 25.8% were degenerated or did not show detectable chromatin. After IVM, the proportion of oocytes resuming meiosis increased significantly (from 16.7% versus 42.7%; P < 0.05), and 9.1% of all oocytes had reached TI or MII. The isolated cumulus–oocyte complexes in Group II began creating follicle-like structures following 24 h of growth culture (7.1%). The proportion of these structures reached 50.8% on days 2–3, and then gradually decreased due to degeneration. On day 10 only 5.8% of cumulus–oocyte complexes were classified as intact. Of the cumulus intact oocytes recovered from the newly created follicle-like structures at 7–10 days, 54.7% were in the germinal vesicle stage, 31.0% underwent germinal vesicle breakdown, 14.3% were
* Corresponding author. Tel.: +49 38208 68754; fax: +49 3820 868752. E-mail address: [email protected] (H. Alm). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.08.014
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degenerated or the chromatin configuration was not detectable. After 24 h of IVM, 67.6% of oocytes had resumed meiosis, and 21.6% of all oocytes had reached TI and MII. These results show that isolated early follicles and cumulus–oocyte complexes from intact early antral follicles can grow in culture and can develop meiotic competence. # 2005 Elsevier Inc. All rights reserved. Keywords: Bovine; Early antral follicles; Culture; IVM
1. Introduction The mammalian ovary contains a huge stock of resting follicles. Each cycle, a number of these follicles is activated to enter the growth phase characterized both by proliferation of granulosa cells and by increase in size of the oocyte [1]. However, during in vivo growth most of these follicles gradually become atretic. In the ovary, there is a greater number of early antral follicles than follicles at more advanced growth stages. The large store of these small follicles creates a potential source of oocytes for in vitro embryo production. Methods allowing the use of these oocytes for in vitro embryo production before they become atretic would enable better utilization of female reproductive potential. Many culture systems have been used to demonstrate that oocytes grow to full size from preantral follicles in mice [2–6], rats [7] and hamster [8]. A live mouse pup was obtained from the culture of oocyte-granulosa cell complexes isolated from neonatal primordial follicles using a complex two-step culture system [9]. However, it has been more difficult to establish a complete in vitro system for preantral follicles obtained from domestic animals. Harada et al. [10] reported that 70% of bovine oocytes isolated from early antral follicles 0.5–0.7 mm in diameter showed normal morphology after 8 days of culture, and increased in diameter (from 95.9 2.8 to 117.7 9.7 mm). The overall maturation potential (MII) was 7 and 11% after 7 and 11 days of culture, respectively. However, using a similar follicle culture system, Miyano et al. [11] reported that only 5% of the oocytes showed developmental competence to the blastocyst stage after IVF. Success of culture of preantral follicles appears to differ by species: when preantral follicles from prepubertal sheep were cultured, only 4–5% of oocytes from these follicles reached metaphase II after subsequent in vitro maturation [12]; however, porcine preantral follicles (200–310 mm in diameter) have been cultured to the antral stage, and after culture for 4 days, 51% of oocytes from these follicles were capable of reaching MII after subsequent IVM [13]. We have previously been shown that late preantral and early antral follicles isolated from bovine ovaries can grow and survive in vitro [14,15]. After 14 days of intact follicle culture, meiotic arrest was preserved in 71.9% of enclosed oocytes. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated immediately after follicle dissection and those cultured in the intact follicle for 6, 8, 11 or 14 days [15]. However, the meiotic competence of these oocytes was not evaluated. The purpose of this study was to characterize meiotic competence of oocytes from cultured early antral bovine ovarian follicles.
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2. Materials and methods The methods for follicle recovery were based on those described in our previous experiments [14,15]. Briefly, ovaries were collected from heifers and cows at local abattoirs and were transported to the laboratory within 2 h. The ovaries were washed 4 times in PBS supplemented with kanamycin (0.075 g/l), and thin cortical slices (approximately 1.0 mm of thickness) were cut from the ovarian surface with a razor blade. The slices were placed for 2 h maximum in a HEPES-buffered medium TCM 199 containing 5% FCS (Sigma) and kanamycin (80 mg/ml), at room temperature. Individual early antral follicles with diameter 0.2–0.7 mm were manually isolated by microdissection of the ovarian slices using fine sharp needles (26 G) under a stereomicroscope. Manipulation for follicle isolation was carried out over approximately 2 h. Early antral follicles were allotted to two treatments: Group I follicles (0.2–0.5 mm diameter) were subdivided into follicle size classes of <225, 225–325, 325–425, 425–525, and >525 mm, and were cultured intact. In Group II follicles (0.4–0.7 mm), cumulus– oocyte complexes with pieces of parietal granulosa (COCGs) were dissected out using the technique presented by Yamamoto et al. [16]. Both intact follicles (Group I) and isolated COCGs (Group II) were embedded in a collagen gel matrix [17]. The matrix was prepared by mixing rat collagen gel solution, 10 times concentrated TCM 199 and sodium bicarbonate solution in a ratio of 7:1:2 (v/v/v). In standard methods for embedding culture objects collagen gel or agar is mainly recommended [18,19], which covers the whole surface of a Petri dish. To compare different volumes of gel for embedding, COCGs were embedded either in microdrops of gel (5 ml + COCG + 5 ml) put in a petri (35 mm in diameter) dish or in a matrix of gel distributed over the whole surface of the petri dish (300 ml + 12 COCG + 200 ml). About 12 COCGs were embedded into the gel. In the case of microdrops, 12 individual drops (5 ml) were placed in the dish, and after placing of one COCG to each drop a second drop of gel was covering the COCG. Immediately after the collagen gelled (12 min at 38 8C under 5% CO2 in air) 2 ml of culture medium was added to the dish. The cultures were fed every 48 h by removing half of the culture medium and adding the same amount of fresh medium. All cultures were kept in an incubator maintained at 38 8C under an atmosphere of 5% CO2 in air. Embedded tissue was cultured in TCM 199 (Earle’s salt) with 3% BSA, ITS (insulin 6.25 mg/ml, transferrin 6.25 mg/ml and sodium selenite 6.25 ng/ml), 100 mg/ml kanamycin, 0.23 mM sodium pyruvate, 2 mM L-glutamine and 4 mM hypoxathine [20]. This culture system allows most follicles to maintain a three-dimensional structure with the presence of a theca layer and basement membrane surrounding the granulosa cells throughout the entire culture period. Intact follicles were cultured for 14 days while COCGs were cultured for 7–10 days. The follicle diameters were measured (n = 111) in Group I follicles at the beginning of culture, on day 6 of culture and at the end of culture (day 14) by using an ocular micrometer on an inverted microscope. Further follicles (n = 194) were cultured for 14 days without frequent measurement. The day the follicles and COCGs were recovered was designated day 0. Oocytes diameter was measured after isolation from the cultured follicle in Group I (n = 66) or from the COCGs in Group II (n = 42) before IVM.
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After the growth culture, cumulus–oocyte complexes (COCs) were recovered from the gel both from follicles and from COCGs. Oocytes that had partially lost the cumulus during growth culture or that were denuded of cumulus were fixed immediately after recovery for evaluation of chromatin configuration; denuded oocytes that showed cytoplasmic fragmentation or shrunken cytoplasm were considered to be degenerated and not evaluated for chromatin configuration. Complexes showing normal morphology were either fixed or used for IVM. They were cultured individually in 10 ml drops of standard IVM medium, i.e. TCM 199 (Earle’s salt) with 20% FCS and 5 mg/ml FSH under oil for an additional 24 h. To evaluate the chromatin configuration in the oocytes, COCs were denuded from the cumulus cells by gentle pipetting with a thin glass pipette, and the oocytes were fixed in buffered formol saline, stained with Hoechst 33258 (2.5 mg/ml) and examined under a fluorescence microscope (wave length 410 nm).
3. Statistics All data except that for follicle growth were evaluated by x2, and all results with P < 0.05 were considered statistically significant. All experiments were repeated 10 times. The data for follicle growth are expressed as the LSM S.E. All data were examined by one-way analysis of variance (ANOVA), using the GLM procedure in SAS/STAT software (SAS Institute Inc. [21]) of the SAS System for Windows (release 8.02). Follicle growth characters (differences in follicle sizes between day 1 to day 6, day 1 to day 14 and day 6 to day 14) were the dependent variables in the ANOVA-model and the fixed classification variable was the effect of follicle size in 5 classes (<225, 225–325, 325–425, 425–525, >525). Additionally, all pair wise differences between the LS-means (LSM) were tested using the PDIFF-option in the LSMEANS-statement of proc GLM.
4. Results 4.1. Group I: intact follicles Altogether 357 early antral follicles were isolated (Fig. 1A), and cultured for 14 days. The diameter of 111 randomly selected follicles of different size groups was measured over the period of culture. The mean diameters of the follicles at the beginning and at the end of growth culture are shown in Table 1. Follicle diameter in all size groups increased significantly in both periods of culture (day 1 to 6 and day 6 to 14). Except for the <225 mm group, a greater rate of growth was seen (as expressed in absolute mm of follicle size) during the first 6 days of culture than from day 6 to day 14 (Table 2). Comparisons of the LS-means in the columns of Table 2 show the effect of the initial follicle sizes (classes) on follicular growth. During the first 6 days of culture the increase in diameter size was significantly higher in follicles with initial diameters between 325 and 425 mm (P < 0.01), 425 and 525 mm (P < 0.01) and larger than 525 mm (P < 0.001) in comparison with follicles having initial
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Fig. 1. Morphology of early antral follicles and cumulus–oocyte-complexes with parietal granulose (COCGs). (A) Freshly dissected very early antral follicle at a starting size of 220 mm. (B) Antral follicle with increased antrum on day 14 of culture (starting size 340 mm; size after culture 533 mm). (C) COCG isolated from a follicle with a diameter of 0.5 mm (before culture). (D) ‘‘New formed follicle’’ 24 h after culture of COCG. (E) Mature oocyte with extruded polar body. The oocyte was obtained from a 14-days cultured follicle (size at recovery 390 mm; size after 14 days of culture 550 mm) and after subsequent IVM for 24 h (bars = 100 mm).
diameters less than 225 mm. In the second period of culture (6–14 days) only the growth in size of follicles with initial diameters between 425 and 525 mm was significantly higher than the growth of follicles with initial diameters between 225 and 325 (P < 0.05). Considering the whole period of culture (1–14 days) the increase in diameter size was significantly higher in the classes 425–525 mm (D: P < 0.0001; F: P < 0.0001) and >
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Table 1 Follicle diameter depending on the duration of the in vitro culture Follicle size classes (mm)
Evaluated follicles (n)
Follicle size day 1 (mm) LSM S.E.
Follicle size day 6 (mm) LSM S.E.
Follicle size day 14 (mm) LSM S.E.
<225 225–325 325–425 425–525 >525
26 30 19 20 16
178.4 7.6 264.1 7.1 386.7 8.9 474.4 8.7 616.7 9.7
204.2 16.1 317.7 16.5 500.3 22.8 590.2 22.0 756.2 22.0
246.9 18.9 348.5 17.6 525.2 24.8 707.6 25.7 806.2 34.0
Follicle diameter in all size groups increased significantly in both periods of culture (days 1–6 and days 6–14). Except for the <225 mm group, a greater rate of growth was seen (as expressed in absolute mm of follicle size) during the first 6 days of culture than from day 6 to day 14 (Table 2).
525 mm (E: P < 0.01; G: P < 0.05) compared with class < 225 mm (D, E) or with class 225–325 mm (F, G). After 14 days of follicle culture, all follicles [n = 305 (261 intact, Fig. 1B, and 44 ‘‘broken’’ with extrusion of oocytes during culture)] were opened. Forty-two percent (n = 129) of all the recovered oocytes were considered to be degenerated of which 44 oocytes were from the broken follicles while 85 oocytes were from the intact follicles. These oocytes were not included and evaluated. One hundred seventy-six oocytes (67.4%) isolated from the intact follicles (n = 261) had a complete cumulus of five or more layers or partially lost cumulus (at least 3 layers of cumulus), and were considered as intact COCs. Of these, 66 oocytes were evaluated immediately after recovery from the cultured follicle. The mean diameter of these oocytes after follicle culture was 105.4 2.4 mm. The remained 110 oocytes having intact cumuli were used for IVM. In oocytes fixed directly after recovery from the cultured follicles 57.6% remained in GV stage, 16.7% underwent GVBD and 16.7% were degenerated. No oocytes had progressed to MII. In Table 2 Follicle growth rate in different culture periods Follicle size classes (mm)
Evaluated follilcles (n)
<225 225–325 325–425 425–525 >525
26 30 19 20 16
ABCDEFGH
Differences in follicle size between Day 1 and day 6 (mm) LSM S.E. (n)
Day 1 and day 14 (mm) LSM S.E. (n)
Day 6 and day 14 (mm) LSM S.E. (n)
25.8ABC 14.6 (26) 58.9* 14.9 (25) 108.4*A 20.6 (13) 109.2*B 19.9 (14) 127.7*C 19.9 (14)
68.5*DE 17.9 (26) 84.4*FG 16.7 (30) 136.2* 23.6 (15) 229.1*DF 24.4 (14) 195.6*EG 32.3 (8)
42.7* 11.3 (26) 27.6*H 11.5 (25) 37.5** 19.1 (9) 98.8*H 20.3 (8) 74.0* 23.4 (8)
equal capital superscript letters: significant different growth between the indicated follicle size classes (P-values in the text). * P < 0.01: significant follicular growth (test of differences vs. 0 within each culture period and follicle size class). ** P < 0.05: significant follicular growth (test of differences vs. 0 within each culture period and follicle size class).
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Table 3 Chromatin configuration in bovine oocytes from intact COCs from freshly isolated follicles, from after culture of isolated follicles (Group I) for 14 days and after additional IVM (n = 228) Time of evaluation
Freshly isolated, without follicle culture After follicle growth After IVM
n
Chromatin configuration (%) Immature
GVBD
52
76.9
9.6 a
66 110
57.6 47.3
16.7a 33.6b
Mature (TI/MII)
Degenerated
No chromatin
0
13.5
0
0 9.1
16.7 5.5
9.1 4.5
Within the columns, values with different superscripts differ significantly; P < 0.02.
oocytes subjected to IVM, after 24 h culture 33.6% were seen to undergo GVBD, and 9.1% had matured to MII (Table 3). To be sure that the culture of follicles for 14 days and of COCGs for 7 days led to achieve developmental potential of the enclosed COCs, we additionally cultured freshly harvested bovine COCs from follicles between 0.4 and 0.7 mm in diameter directly after recovery. No meiotic competence was observed. Seventy-seven percent (40 oocytes) were in germinal vesicle stage after 24 h of IVM. Furthermore, 13.5% were degenerated and 9.5% were in early diakinesis stage. That proves that cumulus enclosed oocytes, both in cultured intact follicles and in the new created follicle-like structures, could grow in our system and develop to oocytes that were able to reach metaphase II after subsequent IVM. 4.2. Group II: isolated COCGs In Group II, 642 COCGs were used for culture (Fig. 1C). Follicle-like structures started to form after 24 h culture in 7.1% of COCGs (Fig. 1D). As the culture time increased, the proportion of these structures increased to 50.8% on day 2–3 of culture. Then these structures started to spread into the gel, and the proportion of intact structures lowered to 30.4% up to days 5–7. After that time most of the follicle-like structures busted and degenerated, and only 5.8% remained intact by day 10. As the newly created follicle-like structures appeared to degenerate after growth culture longer than 7 days, we decided to reduce the duration of growth culture up to day 7 only. Of the originally utilized oocytes 387 had lost most of the cumulus during growth culture and/or showed degenerated cytoplasm, and were, therefore, not considered to be suitable for IVM. Chromatin evaluation of these oocytes showed that 23.5% of the oocytes were in GV-stage, 40.8% had undergone GVBD (mainly early diakinesis), 1.8% were in MII and 33.8% were degenerated or no detectable chromatin structures were observed. Forty-two oocytes, which had intact cumulus, were also fixed for immediate evaluation. These oocytes showed 54.7% GV-stages, 31.0% GVBD and 14.3% degenerated chromatin. The oocyte diameter (n = 42) after COCGs growth was 139.9 2.2 mm. Of the 213 COCs subjected to IVM, 67.6% resumed meiosis. Overall, 21.6% of these oocytes reached telophase I and/or metaphase II after 24 h IVM culture (Table 4, Fig. 1E). The morphology of bovine COCs originating from in vitro growth culture was influenced by the embedding method. The modified method of embedding COCGs in
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Table 4 Chromatin configuration in bovine oocytes after growing in COCGs (Group II) for 7 days and additional 24 h of IVM Time of evaluation
n
After growth culture* After growth culture** After IVM of compact COCs
387 42 213
Chromatin configuration (%) Immature (GV)
GVBD
Mature (TI/MII)
Degenerated/no chromatin
23.5a 54.7b 15.0c
40.8 31.0 46.0
1.8 a 0 21.6b
33.8a 14.3b 17.3b
*
All oocytes with few cumulus and denuded oocytes, values with different superscripts differ significantly; P < 0.01. ** Compact COCs, within the columns, values with different superscripts differ significantly; P < 0.01.
microdrops of collagen gel was associated with significant increase of percentage of oocytes with intact cumuli after 7 days culture in vitro (Table 5). There was no difference in the capacity of cumulus-intact COCs to reach MII between embedding methods.
5. Discussion Biotechnology techniques depend on the predictable production of fully developed oocytes. Currently, their availability is limited by the small number of antral follicles present in the ovaries. An alternative option, i.e. to use follicles, which are not developmentally competent at the time the ovaries are recovered, relies on the possibility of obtaining mature oocytes from in vitro cultured small follicles. The birth of offspring derived from primordial and preantral follicles cultured entirely in vitro has been reported only for the mice [4,22]. In large animals, Newton et al. [23] analyzed the capacity of enzymatically isolated sheep COCs to form antra and to release estradiol release, but this group did not provide information about oocyte meiotic or developmental competence. The culture of early antral follicles as well as the culture of COCGs embedded on collagen gel allowed follicles to develop without losing their normal three-dimensional structure. Similar techniques were used by Gomes et al. [24] and Itoh et al. [25] with mouse Table 5 The quality of bovine COCs originating from 7 day in vitro growth culture in relation to method of their embedding in collagen gel Embedding method
No. of cultured COCGs
No. (%) of COCs
Telo I/Meta II no. (%)
With intact cumulus Collagen gel distributed on the whole surface of Petri dish Microdrops of collagen gel
68
24 (35.3)a
5 (20.8)
87
55 (63.2)b
15 (27.3)
Within the columns, values with different superscripts differ significantly; P < 0.01.
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and bovine preantral follicles. Gomes et al. [24] showed that the flat/adhesive culture of mouse preantral follicles led to distortion of follicular morphology and high rates of spontaneous follicle disruption, whereas three-dimensional collagen gel environments are able to maintain follicular structure with an in vivo-like basal lamina architecture, minimizing spontaneous disruption. In the present study follicles of all initial sizes increased in the diameter during in vitro culture. The growth rate was higher at the beginning of culture (day 1 to day 6) and a reduction of these rates was noticed at the end of culture (up to day 14) for all follicles except those with the smallest diameter (225 mm). Follicles in this group grew more in the second half of the culture period than did the other groups. A higher growth rate at the beginning of culture (after 5 days) and a reduction of these rate at the end of culture (after 10 days) was also observed by Saha et al. [26], working in the bovine; however, these workers cultured preantral follicles with a diameter of 100 mm. Isolated preantral follicles from sheep grew in vitro from a mean diameter of approximately 170 mm to a final diameter of approximately 300–310 mm after 10 days of culture [27]. It was reported that 78% of the follicles were intact and 56% contained healthy COCs. Extrusion of oocytes during culture, which represents a sign of follicle degeneration, was about 5%. In the present study we observed 14.3% ‘‘broken’’ follicles with degenerated oocytes, and the intact follicles (85.6%) contained 67.4% compact COCs. A rapid increase in follicle diameter of bovine preantral follicles during the first week of culture and a slowed growth rate after 8–10 days of culture was observed also by other authors [28]. Oocyte diameter also increased with increasing duration of culture. In our previous experiments [15] bovine oocytes increased from an average diameter of 80.0 9.5 mm to reach 97.9 4.1 mm after 14 days of follicles culture. In the present experiment, the oocytes reached a diameter of 105.4 2.4 mm after 14 days of culture. Increase of oocyte diameter during follicle culture over 14 days was obtained also in other studies (from 55.8 0.8 to 65.7 1.8 mm), but the initial diameter of the follicles was smaller [28]. Similarly, sheep oocytes increased their diameter from 75 5 to 125 2 mm during follicles culture; the in vivo grown oocyte (control) had a diameter of 140 6 mm [27]. The method of embedding COCGs in collagen gel influenced the quality of the enclosed COCs. Generally, the use of collagen gel matrix helps to prevent migration of granulosa cells out of the follicle-like structures created by COCGs. The proportion of COCs having intact cumuli was significantly higher using microdrops of gel than using gel matrix distributed on the whole surface of Petri dish. This higher proportion may be a result of better culture conditions in individual collagen gel droplets. The culture of individual COCGs in a small volume of collagen gel may contribute to the formation of a microenvironment capable of sustaining oocytes and granulosa cells survival. In the present study, we used hypoxanthine-supplemented medium as recommended by Harada et al. [10]. Hypoxanthine is a meiotic-arresting factor present naturally in the follicular fluid of pig and mouse [29,30]. It maintains the association between the mouse oocyte and surrounding granulosa cells in vitro [31] and it seems that the cells exert an inhibitory action on resumption of meiosis in the oocyte through this association. Hypoxanthine was also found to inhibit spreading of the cells, maintain granulosa cell contact with oocytes, and prevented GVBD in mouse oocytes [31]. Though, the
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physiological role of hypoxanthine with respect to the bovine oocyte is obscure because bovine follicular fluid does not contain levels of hypoxanthine comparable to that in other animals [32]. It has been reported that the number of morphologically normal oocytes increases when bovine preantral follicles were cultured in hypoxanthine-supplemented medium [33]. The cumulus–oocyte complexes with pieces of parietal granulosa started to create follicle-like structures after as little as 24 h of culture, and about 50% showed this structure after 3 days of culture. This is similar to the findings of Harada et al. [10] who observed these follicle-like structures up to day 7 of culture. However, in our study we observed a decrease of the portion of the follicle-like structures and a loss of cumulus contact during further growth culture. These discrepancies in our results are difficult to explain. It might be connected with the starting material (for example atretic follicles) from where we isolated the COCGs. Although almost 60% of the cultured COCGs had lost their cumulus cell after 7 days, but not all of the oocytes were degenerated. About 64% were in GV-stage or GVBD (mainly diakinesis), and 33.8% showed degenerated or no chromatin. On the other hand, these oocytes with compact cumulus showed more than 2-times lower degeneration rate in comparison to the denuded oocytes (14.3% when evaluated immediately after growth culture and 17.3% when evaluated following growth culture and IVM). We evaluated the quality of in vitro grown oocytes by analyzing chromatin configuration at the time of recovery of the follicle (Group I) from growth culture and after 24 h of IVM. All of the oocytes having intact cumuli after being isolated from cultured early antral follicles showed normal morphology, and 57.6% of them were at the GV stage, while most of the denuded oocytes were degenerated (shrunken cytoplasm) or had resumed meiosis. Although 42.7% of the in vitro matured oocytes underwent GVBD only 9.1% out of all oocytes reached TI/MII. The comparatively low maturation capacity and the small diameter of the oocytes after follicle culture, 105.4 2.4 mm (normal is 130 mm), both might indicate the suboptimal oocyte development in the present culture conditions. Meiotic competence of oocytes from follicles of different sizes has been investigated in several species. Although oocytes from small follicles can undergo GVBD and resume meiosis, very few progress to metaphase II. Fuhrer et al. [34] reported that that 1.4% of bovine oocytes from follicles <0.9 mm matured to MII, and 48% of oocytes from follicles 2 to 8 mm reached MII following IVM. In pig oocytes from <0.7 mm follicles more than 80% remained in the GV stage. As follicle size increased the percentage of oocytes acquiring the comoetence to mature in vitro also increased [35]. That the size of oocytes obtained from follicles <1 mm influenced the maturation was currently described by Hirao et al. [36]. None of the oocytes with a diameter <100 mm reached metaphase II stage after IVM. On the other hand, oocytes >120 mm reached metaphase II. Fair et al. [37] demonstrated a relationship between oocyte size and acquirement of the ability to complete nuclear maturation to MII and showed that most oocytes <110 mm were transcriptionally active, while oocytes >110 mm did not display transcription. Combelles et al. [38] demonstrated in human a relationship between oocyte size and GV modeling. When bovine COCGs from small antral follicles were embedded in collagen gel for 7 days, 255/642 (40%) COCs were considered to be cumulus-intact after culture. This is similar to the 51% (88/173) cumulus intact oocytes reported by Hirao et al. [36] and lower
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than 70% (23/33) reported by Harada et al. [10]. When we evaluated cumulus-intact oocytes immediately after recovery from growth culture, 54.7% remained in the GV stage. A similar proportion of GV-stage oocytes (45%) was observed by Harada et al. [10] in cumulus-enclosed oocytes after 8 days COCG growth culture. However, only 17% of the oocytes cultured for 7 days and then subjected to IVM underwent GVBD in the study of Harada et al. [10] in comparison to 68% obtained in our study. The rate of maturation to MII was also higher in our study (22%) than in the study of Harada et al. (7%) [10]. Similar low portion of MII stages (4–5%) were achieved in sheep oocytes although 20–43% of the cultured oocytes underwent GVBD [12]. The increased efficiency of maturation in our study could be due culture medium. In the present study, we demonstrated that both isolated follicles with a diameter between 0.2 and 0.5 mm, and cumulus–oocyte complexes with pieces of parietal granulosa isolated from follicles 0.4–0.7 mm in diameter can grow during culture, and the enclosed oocytes can achieve meiotic competence. It is clear that throughout oocyte development in vivo, follicle cell support is fundamental to provide the germ cell with nutrients and growth regulators to ensure progression through the protracted growth phase. The best option available for the complete growth and maturation of oocytes in vitro is to develop an extended multistage culture strategy, which will provide a complex support that closely resembles the ovary in vivo.
Acknowledgments The authors thank Prof. Dr. Katrin Hinrichs, Texas A&M University, for the review of the manuscript. Research was supported by the state committee for scientific research as a solicited project PBZ-KBN-084/PO6/2002 (Poland), and by the Ministry of Agriculture (BMVEL, Germany).
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[9] Eppig JJ, O’Brien MJ. Development in vitro of mouse oocytes from primordial follicles. Biol Reprod 1996;54:197–207. [10] Harada M, Miyano T, Matsumura K, Osaki S, Miyake M, Kato S. Bovine oocytes from early antral follicles grow to meiotic competence in vitro: effect of FSH and hypoxanthine. Theriogenology 1997;48:743–55. [11] Miyano T, Ozaki S, Matsumura K, Miyake M, Kato S, Yamamoto K. In vitro growth, fertilization and development of bovine oocytes from early antral follicles. In: Lauria A, Gandolfi F, Enne G, Gianaroli L, editors. Proceedings of the Serono Symp, Gametes: Development and Function; 1998. p. 575. [12] Cecconi S, Barboni B, Coccia M, Mattioli M. In vitro development of sheep preantral follicles. Biol Reprod 1999;60:594–601. [13] Wu J, Carrell DT, Wilcox AL. Development of in-vitro matured oocytes from porcine preantral follicles following intracytoplasmic sperm injection. Biol Reprod 2001;65:1579–85. [14] Ka˛tska L, Ryn´ska B. The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes. Theriogenology 1998;50:213–22. [15] Ka˛tska L, Alm H, Ryn´ska B. Nuclear configuration of bovine oocytes derived from fresh and in vitrocultured preantral and early antral ovarian follicles. Theriogenology 2000;54:247–60. [16] Yamamoto K, Otoi T, Koyama N, Horikita N, Tachikawa S, Miyano T. Development to live young bovine small oocytes after growing, maturation and fertilization in vitro. Theriogenology 1999;52:81–9. [17] Montesano R, Orci L, Vassalli P. In vitro rapid organization of endothelial cells into capillary-like network is promoted by collagen matrices. J Cell Biol 1983;97:1648–52. [18] Telfer EE. In vitro models for oocyte development. Theriogenology 1998;49:451–60. [19] Wandji SA, Eppig JJ, Fortune JE. FSH and growth factors affect the growth and endocrine function in vitro of granulose cells of bovine preantral follicles. Theriogenology 1996;45:817–32. [20] Alm H, Katska L. Survival and meiotic competence of bovine COCs originating from early antral ovarian follicles. Reprod Dom Anim 2002;37:223 [abstract]. [21] SAS Institute Inc., SAS ISTAT1 User’s Guide Version 6, vol. 2. 4th ed. Cary, NC: SAS Institute Inc.; 1999. [22] O’Brien MJ, Pendola JK, Eppig JJ. A revised protocol for on vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol Reprod 2003;68:1682–6. [23] Newton H, Picton H, Gosden RG. In vitro growth of oocytes-granulosa cell complexes isolated from cryopreserved ovine tissue. J Reprod Fertil 1999;115:141–50. [24] Gomes JE, Correia SC, Gouveia-Oliveira A, Cidadao AJ, Plancha CE. Three-dimensional environments preserve extracellular matrix compartments of ovarian follicles and increase FSH-dependent growth. Mol Reprod Dev 1999;54:163–72. [25] Itoh T, Kacchi M, Abe H, Sendai Y, Hoshi H. Growth, antrum formation, and estradiol production of bovine preantral follicles cultured in a serum-free medium. Biol Reprod 2002;67:1099–105. [26] Saha S, Shimizu M, Geshi M, Izaike Y. In vitro culture of bovine preatral follicles. Anim Reprod Sci 2000;63:27–39. [27] Cecconi S, Capacchietti G, Russo V, Berardinell P, Mattioli M. In vitro growth of preantral follicles isolated from cryopreserved ovine ovarian tissue. Biol Reprod 2004;70:12–7. [28] Gutierrez CG, Ralph JH, Telfer EE, Wilmut I, Webb R. Growth and antrum formation of bovine preantral follicles in long-term culture in vitro. Biol Reprod 2000;62:1322–8. [29] Downs SM, Coleman DL, Ward-Bailay PF, Eppig JJ. Hypoxanthine is the principal inhibitor of murine oocyte maturation in a low molecular weight fraction of porcine follicular fluid. Proc Natl Acad Sci USA 1985;82:454–8. [30] Eppig JJ, Ward-Bailay PF, Coleman DL. Hypoxanthine and adenosine in murine ovarian follicular fluid: concentrations and activity in maintaining oocyte meiotic arrest. Biol Reprod 1985;33:1041–9. [31] Eppig JJ, Downs SM. The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotripin-induced oocyte maturation. Dev Biol 1987;119:313–21. [32] Eppig JJ, Downs SM. The role of purines in the maintenance of meiotic arrest in mammalian oocytes. In: Haseltine FP, First NL, editors. Meiotic inhibition: molecular control of meiosis. NY: Alan R. Liss Inc.; 1988. p. 103–13. [33] Figueiredo JR, Hulshof SCJ, van den Hurk R, Nusgens B, Bevers MM, Ectors FJ, et al. Preservation of oocytes and granulose cell morphology in bovine preantral follicles cultured in vitro. Theriogenology 1994;41:1333–46.
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[34] Fuhrer F, Mayr B, Schellander K, Kalat M, Schlegel W. Maturation competence and chromatin behavior in growing and fully grown cattle oocytes. J Vet Med 1989;36:285–91. [35] Motlik J, Crozet N, Fulka J. Meiotic competence in vitro of pig oocytes isolated from early antral follicles. J Reprod Fertil 1984;71:323–8. [36] Hirao Y, Itoh T, Shimizu M, Iga K, Aiyagi K, Kobayashi M, et al. In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effect of high polyvenylpyrrolidone concentration in culture medium. Biol Reprod 2004;70:83–91. [37] Fair T, Hyttel P, Greve T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Mol Reprod Dev 1995;42:437–42. [38] Combelles CM, Cekleniak NA, Racowsky C, Albertini DF. Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Hum Reprod 2002;17:1006–16.
Survival and meiotic competence of bovine oocytes originating from early antral ovarian follicles H. Alm a,*, L. Ka˛tska-Ksia˛z˙kiewicz b, B. Ryn´ska b, A. Tuchscherer a a
Forschungsinstitut fu¨r die Biologie Landwirtschaftlicher Nutztiere, 18196 Dummerstorf, Rostock, Germany Department of Biotechnology of Animal Reproduction, National Research Institute of Animal Production, Balice, Krakow, Poland
b
Received 23 March 2005; received in revised form 29 August 2005; accepted 30 August 2005
Abstract The aim of the present study was to examine the growth and survival in culture, and the subsequent meiotic competence, of bovine oocytes recovered from early antral ovarian follicles. Follicles isolated by microdissection of the ovarian slices were sorted into two size groups: (I) 0.2–0.5 mm diameter; and (II) 0.4–0.7 mm diameter. Group I follicles were cultured intact while in Group II, cumulus–oocyte complexes with pieces of parietal granulosa were dissected from the follicles and cultured. Follicles or cumulus–oocyte complexes with parietal granulose were embedded in collagen gel and cultured in TCM 199 supplemented with 3% BSA and 4 mM hypoxanthine for 14 days (Group I) or 7–10 days (Group II). After this, cumulus–oocyte complexes were recovered from the gel. Oocytes that had lost the majority of the cumulus were fixed immediately after recovery. Cumulus–oocyte complexes showing normal morphology were either fixed immediately or were subjected to IVM for an additional 24 h, and then were fixed. At the end of the growth culture, 57.6% of the compact COCs in Group I follicles were preserved in the GV configuration, 16.7% had resumed meiosis, and 25.8% were degenerated or did not show detectable chromatin. After IVM, the proportion of oocytes resuming meiosis increased significantly (from 16.7% versus 42.7%; P < 0.05), and 9.1% of all oocytes had reached TI or MII. The isolated cumulus–oocyte complexes in Group II began creating follicle-like structures following 24 h of growth culture (7.1%). The proportion of these structures reached 50.8% on days 2–3, and then gradually decreased due to degeneration. On day 10 only 5.8% of cumulus–oocyte complexes were classified as intact. Of the cumulus intact oocytes recovered from the newly created follicle-like structures at 7–10 days, 54.7% were in the germinal vesicle stage, 31.0% underwent germinal vesicle breakdown, 14.3% were
* Corresponding author. Tel.: +49 38208 68754; fax: +49 3820 868752. E-mail address: [email protected] (H. Alm). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.08.014
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degenerated or the chromatin configuration was not detectable. After 24 h of IVM, 67.6% of oocytes had resumed meiosis, and 21.6% of all oocytes had reached TI and MII. These results show that isolated early follicles and cumulus–oocyte complexes from intact early antral follicles can grow in culture and can develop meiotic competence. # 2005 Elsevier Inc. All rights reserved. Keywords: Bovine; Early antral follicles; Culture; IVM
1. Introduction The mammalian ovary contains a huge stock of resting follicles. Each cycle, a number of these follicles is activated to enter the growth phase characterized both by proliferation of granulosa cells and by increase in size of the oocyte [1]. However, during in vivo growth most of these follicles gradually become atretic. In the ovary, there is a greater number of early antral follicles than follicles at more advanced growth stages. The large store of these small follicles creates a potential source of oocytes for in vitro embryo production. Methods allowing the use of these oocytes for in vitro embryo production before they become atretic would enable better utilization of female reproductive potential. Many culture systems have been used to demonstrate that oocytes grow to full size from preantral follicles in mice [2–6], rats [7] and hamster [8]. A live mouse pup was obtained from the culture of oocyte-granulosa cell complexes isolated from neonatal primordial follicles using a complex two-step culture system [9]. However, it has been more difficult to establish a complete in vitro system for preantral follicles obtained from domestic animals. Harada et al. [10] reported that 70% of bovine oocytes isolated from early antral follicles 0.5–0.7 mm in diameter showed normal morphology after 8 days of culture, and increased in diameter (from 95.9 2.8 to 117.7 9.7 mm). The overall maturation potential (MII) was 7 and 11% after 7 and 11 days of culture, respectively. However, using a similar follicle culture system, Miyano et al. [11] reported that only 5% of the oocytes showed developmental competence to the blastocyst stage after IVF. Success of culture of preantral follicles appears to differ by species: when preantral follicles from prepubertal sheep were cultured, only 4–5% of oocytes from these follicles reached metaphase II after subsequent in vitro maturation [12]; however, porcine preantral follicles (200–310 mm in diameter) have been cultured to the antral stage, and after culture for 4 days, 51% of oocytes from these follicles were capable of reaching MII after subsequent IVM [13]. We have previously been shown that late preantral and early antral follicles isolated from bovine ovaries can grow and survive in vitro [14,15]. After 14 days of intact follicle culture, meiotic arrest was preserved in 71.9% of enclosed oocytes. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated immediately after follicle dissection and those cultured in the intact follicle for 6, 8, 11 or 14 days [15]. However, the meiotic competence of these oocytes was not evaluated. The purpose of this study was to characterize meiotic competence of oocytes from cultured early antral bovine ovarian follicles.
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2. Materials and methods The methods for follicle recovery were based on those described in our previous experiments [14,15]. Briefly, ovaries were collected from heifers and cows at local abattoirs and were transported to the laboratory within 2 h. The ovaries were washed 4 times in PBS supplemented with kanamycin (0.075 g/l), and thin cortical slices (approximately 1.0 mm of thickness) were cut from the ovarian surface with a razor blade. The slices were placed for 2 h maximum in a HEPES-buffered medium TCM 199 containing 5% FCS (Sigma) and kanamycin (80 mg/ml), at room temperature. Individual early antral follicles with diameter 0.2–0.7 mm were manually isolated by microdissection of the ovarian slices using fine sharp needles (26 G) under a stereomicroscope. Manipulation for follicle isolation was carried out over approximately 2 h. Early antral follicles were allotted to two treatments: Group I follicles (0.2–0.5 mm diameter) were subdivided into follicle size classes of <225, 225–325, 325–425, 425–525, and >525 mm, and were cultured intact. In Group II follicles (0.4–0.7 mm), cumulus– oocyte complexes with pieces of parietal granulosa (COCGs) were dissected out using the technique presented by Yamamoto et al. [16]. Both intact follicles (Group I) and isolated COCGs (Group II) were embedded in a collagen gel matrix [17]. The matrix was prepared by mixing rat collagen gel solution, 10 times concentrated TCM 199 and sodium bicarbonate solution in a ratio of 7:1:2 (v/v/v). In standard methods for embedding culture objects collagen gel or agar is mainly recommended [18,19], which covers the whole surface of a Petri dish. To compare different volumes of gel for embedding, COCGs were embedded either in microdrops of gel (5 ml + COCG + 5 ml) put in a petri (35 mm in diameter) dish or in a matrix of gel distributed over the whole surface of the petri dish (300 ml + 12 COCG + 200 ml). About 12 COCGs were embedded into the gel. In the case of microdrops, 12 individual drops (5 ml) were placed in the dish, and after placing of one COCG to each drop a second drop of gel was covering the COCG. Immediately after the collagen gelled (12 min at 38 8C under 5% CO2 in air) 2 ml of culture medium was added to the dish. The cultures were fed every 48 h by removing half of the culture medium and adding the same amount of fresh medium. All cultures were kept in an incubator maintained at 38 8C under an atmosphere of 5% CO2 in air. Embedded tissue was cultured in TCM 199 (Earle’s salt) with 3% BSA, ITS (insulin 6.25 mg/ml, transferrin 6.25 mg/ml and sodium selenite 6.25 ng/ml), 100 mg/ml kanamycin, 0.23 mM sodium pyruvate, 2 mM L-glutamine and 4 mM hypoxathine [20]. This culture system allows most follicles to maintain a three-dimensional structure with the presence of a theca layer and basement membrane surrounding the granulosa cells throughout the entire culture period. Intact follicles were cultured for 14 days while COCGs were cultured for 7–10 days. The follicle diameters were measured (n = 111) in Group I follicles at the beginning of culture, on day 6 of culture and at the end of culture (day 14) by using an ocular micrometer on an inverted microscope. Further follicles (n = 194) were cultured for 14 days without frequent measurement. The day the follicles and COCGs were recovered was designated day 0. Oocytes diameter was measured after isolation from the cultured follicle in Group I (n = 66) or from the COCGs in Group II (n = 42) before IVM.
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After the growth culture, cumulus–oocyte complexes (COCs) were recovered from the gel both from follicles and from COCGs. Oocytes that had partially lost the cumulus during growth culture or that were denuded of cumulus were fixed immediately after recovery for evaluation of chromatin configuration; denuded oocytes that showed cytoplasmic fragmentation or shrunken cytoplasm were considered to be degenerated and not evaluated for chromatin configuration. Complexes showing normal morphology were either fixed or used for IVM. They were cultured individually in 10 ml drops of standard IVM medium, i.e. TCM 199 (Earle’s salt) with 20% FCS and 5 mg/ml FSH under oil for an additional 24 h. To evaluate the chromatin configuration in the oocytes, COCs were denuded from the cumulus cells by gentle pipetting with a thin glass pipette, and the oocytes were fixed in buffered formol saline, stained with Hoechst 33258 (2.5 mg/ml) and examined under a fluorescence microscope (wave length 410 nm).
3. Statistics All data except that for follicle growth were evaluated by x2, and all results with P < 0.05 were considered statistically significant. All experiments were repeated 10 times. The data for follicle growth are expressed as the LSM S.E. All data were examined by one-way analysis of variance (ANOVA), using the GLM procedure in SAS/STAT software (SAS Institute Inc. [21]) of the SAS System for Windows (release 8.02). Follicle growth characters (differences in follicle sizes between day 1 to day 6, day 1 to day 14 and day 6 to day 14) were the dependent variables in the ANOVA-model and the fixed classification variable was the effect of follicle size in 5 classes (<225, 225–325, 325–425, 425–525, >525). Additionally, all pair wise differences between the LS-means (LSM) were tested using the PDIFF-option in the LSMEANS-statement of proc GLM.
4. Results 4.1. Group I: intact follicles Altogether 357 early antral follicles were isolated (Fig. 1A), and cultured for 14 days. The diameter of 111 randomly selected follicles of different size groups was measured over the period of culture. The mean diameters of the follicles at the beginning and at the end of growth culture are shown in Table 1. Follicle diameter in all size groups increased significantly in both periods of culture (day 1 to 6 and day 6 to 14). Except for the <225 mm group, a greater rate of growth was seen (as expressed in absolute mm of follicle size) during the first 6 days of culture than from day 6 to day 14 (Table 2). Comparisons of the LS-means in the columns of Table 2 show the effect of the initial follicle sizes (classes) on follicular growth. During the first 6 days of culture the increase in diameter size was significantly higher in follicles with initial diameters between 325 and 425 mm (P < 0.01), 425 and 525 mm (P < 0.01) and larger than 525 mm (P < 0.001) in comparison with follicles having initial
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Fig. 1. Morphology of early antral follicles and cumulus–oocyte-complexes with parietal granulose (COCGs). (A) Freshly dissected very early antral follicle at a starting size of 220 mm. (B) Antral follicle with increased antrum on day 14 of culture (starting size 340 mm; size after culture 533 mm). (C) COCG isolated from a follicle with a diameter of 0.5 mm (before culture). (D) ‘‘New formed follicle’’ 24 h after culture of COCG. (E) Mature oocyte with extruded polar body. The oocyte was obtained from a 14-days cultured follicle (size at recovery 390 mm; size after 14 days of culture 550 mm) and after subsequent IVM for 24 h (bars = 100 mm).
diameters less than 225 mm. In the second period of culture (6–14 days) only the growth in size of follicles with initial diameters between 425 and 525 mm was significantly higher than the growth of follicles with initial diameters between 225 and 325 (P < 0.05). Considering the whole period of culture (1–14 days) the increase in diameter size was significantly higher in the classes 425–525 mm (D: P < 0.0001; F: P < 0.0001) and >
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Table 1 Follicle diameter depending on the duration of the in vitro culture Follicle size classes (mm)
Evaluated follicles (n)
Follicle size day 1 (mm) LSM S.E.
Follicle size day 6 (mm) LSM S.E.
Follicle size day 14 (mm) LSM S.E.
<225 225–325 325–425 425–525 >525
26 30 19 20 16
178.4 7.6 264.1 7.1 386.7 8.9 474.4 8.7 616.7 9.7
204.2 16.1 317.7 16.5 500.3 22.8 590.2 22.0 756.2 22.0
246.9 18.9 348.5 17.6 525.2 24.8 707.6 25.7 806.2 34.0
Follicle diameter in all size groups increased significantly in both periods of culture (days 1–6 and days 6–14). Except for the <225 mm group, a greater rate of growth was seen (as expressed in absolute mm of follicle size) during the first 6 days of culture than from day 6 to day 14 (Table 2).
525 mm (E: P < 0.01; G: P < 0.05) compared with class < 225 mm (D, E) or with class 225–325 mm (F, G). After 14 days of follicle culture, all follicles [n = 305 (261 intact, Fig. 1B, and 44 ‘‘broken’’ with extrusion of oocytes during culture)] were opened. Forty-two percent (n = 129) of all the recovered oocytes were considered to be degenerated of which 44 oocytes were from the broken follicles while 85 oocytes were from the intact follicles. These oocytes were not included and evaluated. One hundred seventy-six oocytes (67.4%) isolated from the intact follicles (n = 261) had a complete cumulus of five or more layers or partially lost cumulus (at least 3 layers of cumulus), and were considered as intact COCs. Of these, 66 oocytes were evaluated immediately after recovery from the cultured follicle. The mean diameter of these oocytes after follicle culture was 105.4 2.4 mm. The remained 110 oocytes having intact cumuli were used for IVM. In oocytes fixed directly after recovery from the cultured follicles 57.6% remained in GV stage, 16.7% underwent GVBD and 16.7% were degenerated. No oocytes had progressed to MII. In Table 2 Follicle growth rate in different culture periods Follicle size classes (mm)
Evaluated follilcles (n)
<225 225–325 325–425 425–525 >525
26 30 19 20 16
ABCDEFGH
Differences in follicle size between Day 1 and day 6 (mm) LSM S.E. (n)
Day 1 and day 14 (mm) LSM S.E. (n)
Day 6 and day 14 (mm) LSM S.E. (n)
25.8ABC 14.6 (26) 58.9* 14.9 (25) 108.4*A 20.6 (13) 109.2*B 19.9 (14) 127.7*C 19.9 (14)
68.5*DE 17.9 (26) 84.4*FG 16.7 (30) 136.2* 23.6 (15) 229.1*DF 24.4 (14) 195.6*EG 32.3 (8)
42.7* 11.3 (26) 27.6*H 11.5 (25) 37.5** 19.1 (9) 98.8*H 20.3 (8) 74.0* 23.4 (8)
equal capital superscript letters: significant different growth between the indicated follicle size classes (P-values in the text). * P < 0.01: significant follicular growth (test of differences vs. 0 within each culture period and follicle size class). ** P < 0.05: significant follicular growth (test of differences vs. 0 within each culture period and follicle size class).
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Table 3 Chromatin configuration in bovine oocytes from intact COCs from freshly isolated follicles, from after culture of isolated follicles (Group I) for 14 days and after additional IVM (n = 228) Time of evaluation
Freshly isolated, without follicle culture After follicle growth After IVM
n
Chromatin configuration (%) Immature
GVBD
52
76.9
9.6 a
66 110
57.6 47.3
16.7a 33.6b
Mature (TI/MII)
Degenerated
No chromatin
0
13.5
0
0 9.1
16.7 5.5
9.1 4.5
Within the columns, values with different superscripts differ significantly; P < 0.02.
oocytes subjected to IVM, after 24 h culture 33.6% were seen to undergo GVBD, and 9.1% had matured to MII (Table 3). To be sure that the culture of follicles for 14 days and of COCGs for 7 days led to achieve developmental potential of the enclosed COCs, we additionally cultured freshly harvested bovine COCs from follicles between 0.4 and 0.7 mm in diameter directly after recovery. No meiotic competence was observed. Seventy-seven percent (40 oocytes) were in germinal vesicle stage after 24 h of IVM. Furthermore, 13.5% were degenerated and 9.5% were in early diakinesis stage. That proves that cumulus enclosed oocytes, both in cultured intact follicles and in the new created follicle-like structures, could grow in our system and develop to oocytes that were able to reach metaphase II after subsequent IVM. 4.2. Group II: isolated COCGs In Group II, 642 COCGs were used for culture (Fig. 1C). Follicle-like structures started to form after 24 h culture in 7.1% of COCGs (Fig. 1D). As the culture time increased, the proportion of these structures increased to 50.8% on day 2–3 of culture. Then these structures started to spread into the gel, and the proportion of intact structures lowered to 30.4% up to days 5–7. After that time most of the follicle-like structures busted and degenerated, and only 5.8% remained intact by day 10. As the newly created follicle-like structures appeared to degenerate after growth culture longer than 7 days, we decided to reduce the duration of growth culture up to day 7 only. Of the originally utilized oocytes 387 had lost most of the cumulus during growth culture and/or showed degenerated cytoplasm, and were, therefore, not considered to be suitable for IVM. Chromatin evaluation of these oocytes showed that 23.5% of the oocytes were in GV-stage, 40.8% had undergone GVBD (mainly early diakinesis), 1.8% were in MII and 33.8% were degenerated or no detectable chromatin structures were observed. Forty-two oocytes, which had intact cumulus, were also fixed for immediate evaluation. These oocytes showed 54.7% GV-stages, 31.0% GVBD and 14.3% degenerated chromatin. The oocyte diameter (n = 42) after COCGs growth was 139.9 2.2 mm. Of the 213 COCs subjected to IVM, 67.6% resumed meiosis. Overall, 21.6% of these oocytes reached telophase I and/or metaphase II after 24 h IVM culture (Table 4, Fig. 1E). The morphology of bovine COCs originating from in vitro growth culture was influenced by the embedding method. The modified method of embedding COCGs in
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Table 4 Chromatin configuration in bovine oocytes after growing in COCGs (Group II) for 7 days and additional 24 h of IVM Time of evaluation
n
After growth culture* After growth culture** After IVM of compact COCs
387 42 213
Chromatin configuration (%) Immature (GV)
GVBD
Mature (TI/MII)
Degenerated/no chromatin
23.5a 54.7b 15.0c
40.8 31.0 46.0
1.8 a 0 21.6b
33.8a 14.3b 17.3b
*
All oocytes with few cumulus and denuded oocytes, values with different superscripts differ significantly; P < 0.01. ** Compact COCs, within the columns, values with different superscripts differ significantly; P < 0.01.
microdrops of collagen gel was associated with significant increase of percentage of oocytes with intact cumuli after 7 days culture in vitro (Table 5). There was no difference in the capacity of cumulus-intact COCs to reach MII between embedding methods.
5. Discussion Biotechnology techniques depend on the predictable production of fully developed oocytes. Currently, their availability is limited by the small number of antral follicles present in the ovaries. An alternative option, i.e. to use follicles, which are not developmentally competent at the time the ovaries are recovered, relies on the possibility of obtaining mature oocytes from in vitro cultured small follicles. The birth of offspring derived from primordial and preantral follicles cultured entirely in vitro has been reported only for the mice [4,22]. In large animals, Newton et al. [23] analyzed the capacity of enzymatically isolated sheep COCs to form antra and to release estradiol release, but this group did not provide information about oocyte meiotic or developmental competence. The culture of early antral follicles as well as the culture of COCGs embedded on collagen gel allowed follicles to develop without losing their normal three-dimensional structure. Similar techniques were used by Gomes et al. [24] and Itoh et al. [25] with mouse Table 5 The quality of bovine COCs originating from 7 day in vitro growth culture in relation to method of their embedding in collagen gel Embedding method
No. of cultured COCGs
No. (%) of COCs
Telo I/Meta II no. (%)
With intact cumulus Collagen gel distributed on the whole surface of Petri dish Microdrops of collagen gel
68
24 (35.3)a
5 (20.8)
87
55 (63.2)b
15 (27.3)
Within the columns, values with different superscripts differ significantly; P < 0.01.
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and bovine preantral follicles. Gomes et al. [24] showed that the flat/adhesive culture of mouse preantral follicles led to distortion of follicular morphology and high rates of spontaneous follicle disruption, whereas three-dimensional collagen gel environments are able to maintain follicular structure with an in vivo-like basal lamina architecture, minimizing spontaneous disruption. In the present study follicles of all initial sizes increased in the diameter during in vitro culture. The growth rate was higher at the beginning of culture (day 1 to day 6) and a reduction of these rates was noticed at the end of culture (up to day 14) for all follicles except those with the smallest diameter (225 mm). Follicles in this group grew more in the second half of the culture period than did the other groups. A higher growth rate at the beginning of culture (after 5 days) and a reduction of these rate at the end of culture (after 10 days) was also observed by Saha et al. [26], working in the bovine; however, these workers cultured preantral follicles with a diameter of 100 mm. Isolated preantral follicles from sheep grew in vitro from a mean diameter of approximately 170 mm to a final diameter of approximately 300–310 mm after 10 days of culture [27]. It was reported that 78% of the follicles were intact and 56% contained healthy COCs. Extrusion of oocytes during culture, which represents a sign of follicle degeneration, was about 5%. In the present study we observed 14.3% ‘‘broken’’ follicles with degenerated oocytes, and the intact follicles (85.6%) contained 67.4% compact COCs. A rapid increase in follicle diameter of bovine preantral follicles during the first week of culture and a slowed growth rate after 8–10 days of culture was observed also by other authors [28]. Oocyte diameter also increased with increasing duration of culture. In our previous experiments [15] bovine oocytes increased from an average diameter of 80.0 9.5 mm to reach 97.9 4.1 mm after 14 days of follicles culture. In the present experiment, the oocytes reached a diameter of 105.4 2.4 mm after 14 days of culture. Increase of oocyte diameter during follicle culture over 14 days was obtained also in other studies (from 55.8 0.8 to 65.7 1.8 mm), but the initial diameter of the follicles was smaller [28]. Similarly, sheep oocytes increased their diameter from 75 5 to 125 2 mm during follicles culture; the in vivo grown oocyte (control) had a diameter of 140 6 mm [27]. The method of embedding COCGs in collagen gel influenced the quality of the enclosed COCs. Generally, the use of collagen gel matrix helps to prevent migration of granulosa cells out of the follicle-like structures created by COCGs. The proportion of COCs having intact cumuli was significantly higher using microdrops of gel than using gel matrix distributed on the whole surface of Petri dish. This higher proportion may be a result of better culture conditions in individual collagen gel droplets. The culture of individual COCGs in a small volume of collagen gel may contribute to the formation of a microenvironment capable of sustaining oocytes and granulosa cells survival. In the present study, we used hypoxanthine-supplemented medium as recommended by Harada et al. [10]. Hypoxanthine is a meiotic-arresting factor present naturally in the follicular fluid of pig and mouse [29,30]. It maintains the association between the mouse oocyte and surrounding granulosa cells in vitro [31] and it seems that the cells exert an inhibitory action on resumption of meiosis in the oocyte through this association. Hypoxanthine was also found to inhibit spreading of the cells, maintain granulosa cell contact with oocytes, and prevented GVBD in mouse oocytes [31]. Though, the
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physiological role of hypoxanthine with respect to the bovine oocyte is obscure because bovine follicular fluid does not contain levels of hypoxanthine comparable to that in other animals [32]. It has been reported that the number of morphologically normal oocytes increases when bovine preantral follicles were cultured in hypoxanthine-supplemented medium [33]. The cumulus–oocyte complexes with pieces of parietal granulosa started to create follicle-like structures after as little as 24 h of culture, and about 50% showed this structure after 3 days of culture. This is similar to the findings of Harada et al. [10] who observed these follicle-like structures up to day 7 of culture. However, in our study we observed a decrease of the portion of the follicle-like structures and a loss of cumulus contact during further growth culture. These discrepancies in our results are difficult to explain. It might be connected with the starting material (for example atretic follicles) from where we isolated the COCGs. Although almost 60% of the cultured COCGs had lost their cumulus cell after 7 days, but not all of the oocytes were degenerated. About 64% were in GV-stage or GVBD (mainly diakinesis), and 33.8% showed degenerated or no chromatin. On the other hand, these oocytes with compact cumulus showed more than 2-times lower degeneration rate in comparison to the denuded oocytes (14.3% when evaluated immediately after growth culture and 17.3% when evaluated following growth culture and IVM). We evaluated the quality of in vitro grown oocytes by analyzing chromatin configuration at the time of recovery of the follicle (Group I) from growth culture and after 24 h of IVM. All of the oocytes having intact cumuli after being isolated from cultured early antral follicles showed normal morphology, and 57.6% of them were at the GV stage, while most of the denuded oocytes were degenerated (shrunken cytoplasm) or had resumed meiosis. Although 42.7% of the in vitro matured oocytes underwent GVBD only 9.1% out of all oocytes reached TI/MII. The comparatively low maturation capacity and the small diameter of the oocytes after follicle culture, 105.4 2.4 mm (normal is 130 mm), both might indicate the suboptimal oocyte development in the present culture conditions. Meiotic competence of oocytes from follicles of different sizes has been investigated in several species. Although oocytes from small follicles can undergo GVBD and resume meiosis, very few progress to metaphase II. Fuhrer et al. [34] reported that that 1.4% of bovine oocytes from follicles <0.9 mm matured to MII, and 48% of oocytes from follicles 2 to 8 mm reached MII following IVM. In pig oocytes from <0.7 mm follicles more than 80% remained in the GV stage. As follicle size increased the percentage of oocytes acquiring the comoetence to mature in vitro also increased [35]. That the size of oocytes obtained from follicles <1 mm influenced the maturation was currently described by Hirao et al. [36]. None of the oocytes with a diameter <100 mm reached metaphase II stage after IVM. On the other hand, oocytes >120 mm reached metaphase II. Fair et al. [37] demonstrated a relationship between oocyte size and acquirement of the ability to complete nuclear maturation to MII and showed that most oocytes <110 mm were transcriptionally active, while oocytes >110 mm did not display transcription. Combelles et al. [38] demonstrated in human a relationship between oocyte size and GV modeling. When bovine COCGs from small antral follicles were embedded in collagen gel for 7 days, 255/642 (40%) COCs were considered to be cumulus-intact after culture. This is similar to the 51% (88/173) cumulus intact oocytes reported by Hirao et al. [36] and lower
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than 70% (23/33) reported by Harada et al. [10]. When we evaluated cumulus-intact oocytes immediately after recovery from growth culture, 54.7% remained in the GV stage. A similar proportion of GV-stage oocytes (45%) was observed by Harada et al. [10] in cumulus-enclosed oocytes after 8 days COCG growth culture. However, only 17% of the oocytes cultured for 7 days and then subjected to IVM underwent GVBD in the study of Harada et al. [10] in comparison to 68% obtained in our study. The rate of maturation to MII was also higher in our study (22%) than in the study of Harada et al. (7%) [10]. Similar low portion of MII stages (4–5%) were achieved in sheep oocytes although 20–43% of the cultured oocytes underwent GVBD [12]. The increased efficiency of maturation in our study could be due culture medium. In the present study, we demonstrated that both isolated follicles with a diameter between 0.2 and 0.5 mm, and cumulus–oocyte complexes with pieces of parietal granulosa isolated from follicles 0.4–0.7 mm in diameter can grow during culture, and the enclosed oocytes can achieve meiotic competence. It is clear that throughout oocyte development in vivo, follicle cell support is fundamental to provide the germ cell with nutrients and growth regulators to ensure progression through the protracted growth phase. The best option available for the complete growth and maturation of oocytes in vitro is to develop an extended multistage culture strategy, which will provide a complex support that closely resembles the ovary in vivo.
Acknowledgments The authors thank Prof. Dr. Katrin Hinrichs, Texas A&M University, for the review of the manuscript. Research was supported by the state committee for scientific research as a solicited project PBZ-KBN-084/PO6/2002 (Poland), and by the Ministry of Agriculture (BMVEL, Germany).
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