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Nuclear configuration of bovine oocytes derived from fresh and in vitro-cultured preantral and early antral ovarian follicles
ELSEVIER
NUCLEAR CONFIGURATION OF BOVINE OOCYTES DERIVED FROM FRESH AND IN VITRO-CULTURED PREANTRAL AND EARLY ANTRAL OVARIAN FOLLICLES L. Klitska,la H. Alm 2 and B. Rynska! IDepartment of Animal Reproduction, National Research Institute of Animal Production, Balice/Krakow, Poland; 2Department of Reproductive Biology, Research Institute for the Biology of Farm Animals, Dummerstorf/Rostock, Germany Received for publication: August 26, 1999 Accepted: May 18, 2000 ABSTRACT The aim of this experiment was to characterize the growth and nuclear configuration of oocytes isolated from late preantral and early antral bovine ovarian follicles immediately after recovery and after the in vitro culture . Individual follicles were isolated by microdissection from slices of the ovarian cortex . Follicles were sorted by diameter into 175 to 224, 225 to 274 and 275 to 325 urn-size classes. The follicles selected for in vitro culture were placed singly into 40 ul. droplets of medium (TCM 199 enriched with FCS, insulin, transferrin , sodium selenite, sodium pyruvate , l-glutarnine, hypoxanthine , FSH and estradiol-Up) and cultured for 6, 8, II, 14 or 17 d. The sizes of follicles and oocytes were related to the duration of culture and gradually increased as culture duration was prolonged. The analysis of the relationship between mean diameters of oocytes at the time of recovery and after the in vitro culture, has shown significant differences after culture lasting 8 d (76.9±9.9 vs. 86.1±1l.lllm; P< 0.05), II d (77.0±9.9 vs. 91.9±17.5 11m; P< 0.01), 14 d (80.0±9.5 vs. 97.9±16.5 11m; P< 0.01) and 17 d (82.6±6.6 vs. 97.2±11.5 11m; P< 0.01). No statistical differences were shown among oocytes in the 5 pre-culture groups (79.5±8.8 ; 76.9±9.9; 77.1±9.9; 80.l±9.5 and 82.6±6.6 urn). Meiotic arrest was preserved in 71.9 % of oocytes in our culture system up to 14 d. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated "fresh" or cultured for 6, 8, II or 14 d. No relationship was observed between the size class of follicles and the frequency of the GV-stage. Prolonging the culture period to 17 d drastically decreased the percentage of oocytes in the GVstage (18.7 %) and increased the percentage of oocytes having premature initiation of meiosis (GVBD; 46.3 %) and degeneration (25.0 %). These results suggest that out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at the GV-stage. @
2000 by Elsevier Science Inc.
Key words: cattle, preantral follicle, in vitro culture, oocyte, germinal vesicle
Acknowledgments The authors thank Dr. K. Hinrichs of the Texas A&M University for linguistic review and critical reading of the manucript, Dr. R. Poehland for the computer processing of the pictures , and M.S. H. Korzonek for statistical help. a Correspondence and reprint requests: Lucyna Katska, National Research Institute of Animal Production, Department of Animal Reproduction , 32-083 Balice/Krakow, Poland. Tel. (48)( 12) 285 6714; e-mail : [email protected] Theriogenology 54:247-260, 2000 Cl2000 Elsevier SCience Inc.
0093·691X!OO/$-see front matter PII: SOO93·691X(OOlOO345-9
248
Theriogenology INTRODUCTION
In cattle, a follicle that has formed an antrum needs 40 d to reach ovulatory size (12). By extrapolation of these data it was estimated that the preantral stage (primary or secondary) follicle requires at least 60 to 80 d to reach maturity. Small preantral follicles (diameter 35 to 100 urn) can survive in culture up to 6 or 7 d, and during this time cell proliferation and follicle growth are observed (1,6,8,17,20,21). However, the quality of oocyte ultrastructure appears to be unsatisfactory not only after culture but even immediately after follicle isolation. Recently, Schotanus et al. (17) and van den Hurk et al. (19) have reported that over 90% bovine preantral follicles (less than 60 urn in diameter), isolated mechanically by a tissue chopper, showed a poor uItrastucture, especially in their oocytes. These follicles were characterized by swollen mitochondria, a wrinkled nuclear membrane, and vacuoles in the cytoplasm of the oocyte or in the cytoplasm of both the granulosa cells and the oocyte. Morphology was considered normal when it resembled that of the follicles in the ovarian slices (17). In contrast with the small follicles, the percentage of ultrastructurally poor large preantral follicles had decreased to 27 % after 5 d of culture, possibly due to betterisolation and culture conditions (19). Recently, Harada et al. (9) reported that bovine oocytes isolated from early antral follicles 0.5-0.7 mm in diameter successfully grew during in vitro culture for 11 d and their diameter increased from 90-99 urn to 108.0 ± 5.3 11m. Some oocytes from follicles of this size, similarly cultured as in the Harada et a1. (9) experiment, were able to reach meiotic competence, fertilization and developmental competence as well (14). We have reported in a previous experiment (II), that the survival time of follicles is related to their initial size at the beginning of culture. The longest period of growth was observed for follicles 275 to 324 um in diameter (i.e, 10.7±5.7; 12.1±6.2 and 12.2±2.7 d, respectively, for culture in supplemented' Menezo B2, TCM 199 enriched with fetal calf serum (FCS), and TCM 199 enriched with steer serum. Survival and growth of some follicles were maintained for 23 d. Moreover, we have shown the positive influence of FSH, estradiol-17 13, hypoxathine, insulin, transferrin, sodium selenite, sodium pyruvate, and l-glutamine on follicle survival and growth of medium supplementation (11). There are now reports on several culture systems that support bovine preantral follicle development for a few days (reviewed by Telfer (18) and van den Hurk et al. (19) but these studies present little information on nuclear configuration of oocytes, especially when they are recovered from late preantral and early antral follicles. During the prolonged culture of follicles, it is essential to know whether the follicle maintains meiotic arrest of the oocyte and how the oocyte survives in the culture. The preliminary results of our previous study (II) have indicated that the morphologically normal structure of oocytes (i.e. germinal vesicle oocyte with compact, dense-cumulus layers and homogenous, intact cytoplasm) was preserved only in approximately 35% of the follicles that survived in vitro culture for more than 2 wk. It has seemed to us, therefore, essential to characterize nuclear configuration of oocytes both immediately after recovery of follicle and after in vitro culture, using late preantral and early antral follicles.
249
Theriogenology MATERIALS AND METHODS
The methods for follicle recovery and culture were based on those described in our previous experiment (11) . Briefly, ovaries were collected from heifers and cows at a local abattoir and were transported to the laboratory within I h. The ovaries were washed 4 times in PBS supplemented with kanarnycins (0.075 gIL), and thin cortical slices « 1.0 mm ofthiekness) were prepared with a razor blade. The slices were stored in a HEPES-buffered medium TCM 199 containing 5% Fcsa and antibiotics, at room temperature. Individual preantral and very early antral follicles were isolated by microdissection of the ovarian slices (teased from adhering stroma) using fine sharp needles (30 g 1/2 inch) fitted to 1mLsyringe barrels or acupuncture needlesb (0.25 diameter) under a stereomicroscope at x 80 magnification. Follicle isolation was carried out over approximately 2 h of manipulation (II). Intact preantral and early antral follicles were sorted by diameter as previously described (II) into 175 to 224, 225 to 274 and 275 to 325 um size classes. The follicles surrounded by an intact basal membrane and possessing clear and compact granulosa cell layers were selected for the experiment. Approximately one-third of selected follicles served as a control; they were ruptured immediately after recovery and isolated oocytes were measured by diameter and fixed for evaluation of nuclear configuration. The follicles used for in vitro culture were randomly distributed across the different times of cultu re. Follicles were individually transferred into droplets of medium in a 60-mm Petri dish C conta ining 14 x 40 ul, droplets, covered with 5 mLof the mineral oil a . On day 2 of the culture, the medium was replenished by removing 20 ~L and replacing it with 20 ul, of fresh medium , and thereafter half of the medium was refreshed every 48 h. The culture medium was composed of TCM 199 with 10% heat-inactivated FCS, ITS (insulin 6.25 ug/rnl., transferrin 6.25 ug/ml, and sodium selenite 6.25 ng/rnl.)«, 0.23 mM sodium pyruvates, 2 mM Lsglutaminee, 2 mM hypoxanthines, FSHa (from human pituitary, 0.05 IU/mL) and estradiol-l Zp'' (l)lg/ mL) (II). Follicles were cultured in an incubator at 38.5°C, 100% humidity and 5% C02 in air, for 6, 8, II, 14 or 17 d. Characterization of the cultured follicles was done on Day 0 by means of a phase-contrast inverted microscope. Follicle growth was monitored daily by measurement of follicular diameter using a precalihrated ocular micrometer on an inverted microscope at x 100 magnification. Follicular survival in culture was considered positive as long as the basal membrane remained intact and no visible signs of degeneration were noticed. Morphological criteria of follicular degeneration after culture included the following signs : follicular outline becoming irregular and dark patches of dead cells appearing within the membrana granulosa, especially around the oocyte . Oocytes were freed from their follicles immediately after recovery ("fresh") and after 6, 8, II, 14 or 17 d of the in vitro culture. Oocyte size assessment was carried out after mechanical removal of the oocytes from their follicles under a stereomicroscope. Follicles were punctured with thin , sharp acupuncture needles until oocytes were released into the manipulation medium . Those oocytes that still showed the presence of follicle cells surrounding the zona pellucid" were gently pipettcd through a narrow glass pipette. Only oocytes free of follicle cells were used for further evaluation. Oocyte quality was recorded immediately at the time of follicle recovery
Sigma Chemical Company, S1. Louis, MO , USA. b Acumedic LTD, London, UK. C Falcon, Becton, Dickinson, USA.
a
Theriogen%gy
250
("fresh") and after 6, 8, II, 14 or 17 d of culture. The evaluation of oocyte quality included the size of the oocyte with or without the zona pellucida and nucleus configuration. Nuclear and chromatin status was evaluated after oocyte had been mounted onto a slide, fixed in acetic ethanol (CH3COOH: C2H50H= I :3), stained with I% aceto-orcein and examined under phasecontrast microscope at x 1000 magnification. Statistical differences in the diameters of the oocyte were analyzed by the Student's t-test. Proportion of oocytes in different stage of meiosis was compared using the Chi-square test. Differences with P<0.05 were considered to be statistically significant. RESULTS A total of 392 bovine follicles with a mean diameter of approximately 250 urn were analyzed. Figure I shows an example of a follicle at the time of recovery that was used for the experiment. During culture, cells originating from the surface of the follicle attached themselves to the dish and proliferated, forming a monolayer surrounding the follicle and attaching it to the dish (Figure 2). By Day II, almost all follicles had become attached to the dish and the follicles reached a "spread appearance" (Figure 3). The size of the antral cavity (indicated by the appearance of gaps within the granulosa cell mass) gradually increased (Figures 3 and 4). More than 50 % of the oocytes freed from their follicles maintained oocyte-granulosa connections (Figure 5). Other oocytes lost connections with the granulosa cells (denuded oocytes), degenerated or completely disappeared while granulosa cells appeared healthy. Denuded oocytes were saved for further evaluation while oocytes with heavy degenerated cytoplasm were discarded. Table I. Classification and characterization of bovine oocytes preantral and early antral ovarian follicles
isolated from
fresh, late
Follicle size classes
Evaluated follicles
Follicle diameter, f.!m
No. (%) of recovered
(urn)
(n)
(mean±SO)
oocytes
(mean ± SO) with zpa without zpa
175-224
14
198.3 ± 14.4
6 (42.9)
85.6 ± 9.5
70.5 ± 3.5
225-274
30
248.7 ± 13.1
21 (70.0)
79.5 ± 8.8
64.0 ± 8.5
304.1 ± 16.2
10 (83.3)
85.9 ± 8.9
69.2 ± 6.3
275-324 12 azp - the zona pellucida
Oocyte diameter, urn
The relationships between follicle diameter before and after culture and oocyte diameter with and without zona pellucida are shown in Tables I and 2. Diameter of both follicles and oocytes was related to the culture period and gradually increased as the culture time was prolonged. The analysis of the relation between mean diameters of the oocyte at the time of recovery and after in vitro culture has shown significant differences after culture lasting 8 d (76.9±9.9 vs. 86.1± 11.1 urn; P< 0.05), II d (77.1±9.8 vs. 91.9± 17.5 urn; p< 0.01), 14 d (80.0±9.5
251
Theriogenology
Figure I. Freshly isolated bovine earlyantral follicle. Scale bar represents 50 /.lID. 00: oocyte; arrow: basal lamina.
-Figure 2. Follicle attached to the culture dish after 8 days of in vitro culture . Scale bar represents 100 ).lID. 00: oocyte; arrow: basal lamina .
252
Theriogenology
Figure3. Follicle reaching "spreadappearance" after 11daysof in vitroculture. Antrum visible as emptypatches withinthe granulosa cell mass. Scalebar represents 100IJ.m. 00: oocyte; A: antrum; arrow: basallamina.
Figure 4. The samefollicle as on Figure 3 but after 17daysof in vitroculture. Increase of antrumclearlyvisible. Scalebar represents 100IJ.m. 00: oocyte; A: antrum.
253
Theriogenology
/
I
Figure 5. Oocyte freed from the follicle after I I days of in vitro culture. Oocyte surrounded by compact and dense cumulus cell layers. Scale bar represents I00 urn, 00: oocyte.
Figure 6. Oocyte in GV stage after 14 days ofin vitro culture. Scale bar represents germinal vesicle; arrow: nucleolus.
1O~.
GV:
I\)
~
Table 2. Characterization of bovine oocytes derived from fresh and in vitro-cultured late preantral and early antral ovarian follicles
Duration Follicles of culture totala/cult. (d) N
Diameters before culture, 11m, mean ± SO, (n) Follicle
Oocyte with Zpb
Oocyte without Zpb
Diameters after culture, 11m, mean ± SD, (n) Follicle
Oocyte with
zr"
Oocyte without Zpb
6
88/58
245.9 ± 54.6
c 76.6 ± 8.8 (28)
c 64.0 ± 8.5 (28)
330.1 ± 76.6
c 79.5 ± 8.8 (30)
8
58/45
244.7 ± 42.7
c 76.9 ± 9.9 (13)
c 64.0 ± 8.5 (13)
373.0 ± 9804
86.1 ± IUd (33)
c 69.5 ± IlA (33)
II
43/22
253.7 ±42.9
c 77.1 ± 9.9 (19)
c 64.0 ± 8.5 (19)
393.4 ± 1l1.8
e 91.9 ± 17.5 (19)
67.9 ± 16f (19)
14
92/35
253.7 ± 40.0
c 80.0 ± 9.5 (52)
c 66.6 ± 7.5 (52)
456.9 ± 114.2
e 97.9 ± 16.5 (35)
d 80.3 ± 21.2 (35)
17
55/39
265.5 ± 40.3
c 82.6 ± 6.6 (14)
c 69.2 ± 6.3 (14)
654.6 ± 172.9
97.2 ±
n.s" (14)
80.3 ± llA (14)
~otal
69.8 ± 9 f (30)
c
no. Include the control (fresh) and the in vitro cultured follicles used for measurement of the initial diameter.
bZp _ zona pellucida.
cod. c:eComparisons were made within the row between diameters of fresh and cultured oocytes, means with different superscripts are significantly different (d p<0.05, c:ep
:;t m
5'
~o
c-
~
Theriogenology
255
vs. 97.9±16.5 um; P< 0.01) and 17 d (82.6±6.6 vs. 97.2±11.5 urn; P< 0.01). No statistical differences were shown in the oocyte diameters among the 5 pre-culture groups (Table 2). Meiotic arrest was maintained in oocytes up to 14 d of in vitro culture. The frequency of the germinal vesicle (OV) stage did not differ significantly between oocytes evaluated as "fresh" and those cultured for 6,8, II and 14 d, being 75.7; 59.5; 60.0; 75.1 and 71.9 %, respectively (Table 3, Figure 6). No relationship was observed between follicle size and frequency of the OV-stage in fresh oocytes and those cultured up to 14 d (Figures 7a to e). However, prolonging the culture period' to 17 d decreased the percentage of oocytes in GV-stage (18.7 %), and increased the incidence of premature meiosis initiation and oocyte degeneration (Table 3 and Figure 7f). A lower percentage of oocytes (41.0 %) was recovered after the culture was prolonged up to 17 d in comparison to those released from the follicles and cultured for 6 to 14 d. Approximately 43 to 83% of the oocytes recovered from uncultured follicles (Table I) and 41 to 91 % of the oocytes from the in vitro cultured follicles (Table 3) were analyzable. The rest of the oocytes that were not used for the microscopic investigation, included those not recovered, absent or lost during manipulations, i.e, broken on isolation, lost during fixation/staining, very tightly covered by cumulus cells' (nuclear configuration undetermined) and/or with completely degenerated cytoplasm t'empty" zona pellucida). DISCUSSION In cattle, oocytes in early antral follicles do not reach their final size of 120 urn until after the antrum has formed (5). The growth period appears to be considerably long. Therefore, a culture system supporting growth of both follicle and oocyte should be developed. Culture efficacy can be characterized by maintenance of a germinal vesicle configuration and a normal oocyte morphology through the period of growth. Defined homogenous oocyte population in GVstage at recovery is essential for effectiveness of the IYMlIVF technique. In our previous experiment (II) on isolation and in vitro culture of late preantral and early antral bovine ovarian follicles, we reported that the total growth, growth rate and survival of follicles of different size were similar in the 2 media (TCM 199 and Menezo B2). However, the culture of follicles in the TCM 199 that was supplemented only with steer serum significantly reduced follicular survival and growth in comparison with follicles cultured in the medium containing other additives such as FSH, ITS, I-glutamine, sodium pyruvate and hypoxanthine. Some late preantral and early antral follicles survived and grew up to 23 d, but their mean growth span did not exceed 10 to 12 d. Even though considerable differences were noticed in the length of time follicles survived in vitro, there were no obvious differences in the follicular growth rate per day of culture and size increase after culture. The highest growth rates of follicles were observed at the beginning of culture and a gradual reduction of these rates during the culture period was noted. The preliminary analysis of nuclear configuration in oocytes freed from follicles that had survived in vitro culture for more than 2 wk has indicated that GY-configuration was preserved only in approximately 35% of the oocytes (II). Therefore, it has seemed to us essential to determine the frequency of GY-configuration in oocytes freed from follicles immediately after their recovery and after several days of the in vitro culture and to establish the relationship between the size of follicle and/or the duration of the in vitro culture and the preservation of OV-configuration.
I\:)
~ (a)
ro)
Control (fresh ooc ytes)
>
*'
l;l, 60.0
60.0 60.0
~ 0
40.0 ' -
~
20.0
~
20.0 · -
-
QO
0.0 175224
125174
225274
275324
>325
d
.
175224
(e)
_I
100.0
I
60.0
II ~
-
-
---
60.0
--
40.0
-
20.0
-
-
-
-
-
-
-
-
225274
275324
follicularsize classes
-
~
40.0
-
;.
20.0 ·
-
-
QO .
.
225274
275324
>325
I
-
~
>325
I
(f)
~
-- -
> ~0
1-
40.0 20.0
.
175224
~
1-
225274
275324
>325
folliculars ize class es
--
-
.
175224
275324
>325
-
.
225274
Mean
17 days ofcuhure 1 00 ~
125174
Mean
-
foll icular size classes
60.0 l;l, ~ 60.0
> ~0
-
-
-
.
125174
Mean
14 days ofcuhure
0.0 175224
-
60.0 60.0
100.0
0.0 125174
-
;
follicular size classe s
I I days of culture
)-
-
.
125174
Mean
-
foll icular sill: classe s
r
8 days ofculture
'OO'1iifij 'OO'iiim ~ ::~ - ==== - -
100.0
~
(c)
6 days ofcuhure
60 0 60 0 ~O
mo QO
Mean
-~l
125174
175224
225274
275324
>325
_
Mean
follicularsize classes
Figure 7. Percentage of oocytes in GV stage. Oocytes isolated from fresh (a) and in vitro (b to f) bovine preantral and early antral follicles. (The initial diameter of follicles was used for classification.)
~
Q)
g.
1 is'
~
:;! (1)
g. Tabl e 3. Nuclear status of oocytes recovered from fresh and in vitro-cultured preantral and early antral bovine ovarian follicles
~o
0-
~ Culture time
Follicles
(d)
(n)
Oocytes recovered
Germinal vesicle
n (%)
Stage of meiosis, No. ( %) of oocytes Germinal vesicle breakdown
Degenerated
a Intact 4 (10.8)
b Atretic 3 (8.1)
c 2 (5.4)
0
56
37(66.1)
28 (75.7)
Total c(IS .9) 7
6
55
37 (67.3)
c 22 (59.5)
SC (2 1.6)
2 (6.4)
6 (16.2)
c 7 (18.9)
8
45
30 (66.7)
18c (60.0)
7c (23.3)
3 (10.0)
4 (13.3)
c 5 (16.7 )
11
22
16 (72.7)
12c (75.1)
3c (18.S)
I (6.3)
2 (12.5)
IC(6.3)
14
35
32 (91.4)
23 c (71.9)
6c (18.8)
4 (12.5)
2 (6.3)
c 3 (9.4 )
17
39
16 (41.0)
3e (18.7)
ge (46.3)
6 (37.5)
3 (IS .S)
d 4 (25.0)
c
a Intact GVBD- oocyte with diakinesis chromat in, the nuclear envelope partially or completely dispersed. b
Atretic GVBD- oocyte with highly condensed, pyknotic chromatin, nuclear envelope partially or completely dispersed .
cod; c:eComparisons were made within the columns ; values with different superscripts are significantly different (c:dp <0.05, c:cp< O.OI, X2 test). I\)
~
258
Theriogenology
The results of the present study showed that a relatively high proportion of fresh late preantral and early antral bovine follicles contained oocytes that appear to have undergone resumption of meiosis. Because of the very small follicle size, this high percentage in GYBD probably represents atresia. Resumption of meiosis may occur spontaneously in atretic ovarian follicles (7,13). Our results demonstrate that late preantral and early antral follicles with a mean diameter of approximately 250 flm increased in size during culture period lasting up to 17 d. As we have already mentioned, during the culture spindle-shaped cells, which probably represented fibroblasts originating from surface of the follicle (the rest of ovarian stroma cells and/or the theca cells), attached themselves to the dish and proliferated, with the formation of a monolayer surrounding and attaching the follicle to the dish. The initial follicular structure was lost and follicles developed a "diffuse appearance". A similar phenomenon was reported by Cortvrindt et al. (2) for in vitro-cultured mouse preantral follicles, but the loss of normal three-dimensional follicular structure did not affect oocyte quality and function of all components, i.e. theca, granulosa and oocyte. In our experiment follicular growth was accompanied by growth of oocytes freed from these follicles after 8 to 17 d of the in vitro culture. A higher rate of oocyte growth comparing to values for fresh, uncultured oocytes was observed after culture lasting longer than 8 d. The oocyte growth was noted also after 17 d of culture, however the quality of oocytes was unsatisfactory. However, it should be pointed out that the oocyte in late preantral or early antral bovine follicle is only occasionally clearly visible under the inverted microscope, therefore measurements of the oocyte diameter before and after culture could not be carried out. For this reason, the calculation of oocyte growth was done by the comparison of mean diameter of the oocytes recovered from the same size class of follicles that were freshly isolated or cultured in vitro. As we have already mentioned, the final arbiter of successful follicle growth in vitro is the quality of the oocyte. Although follicles cultured for 17 d grew, when they were mechanically dissected, most of the oocytes recovered were degenerated or were no longer in meiotic arrest. Morphological analysis of these follicles revealed signs of degeneration. In these follicles, disruption of intercellular contacts or build up of metabolic degradation products may have resulted in loss of the follicle's ability to maintain meiotic arrest, resulting in a decreased rate of the Gv-stage, premature resumption of meiosis in the oocytes and high incidence of oocyte degeneration. Therefore, the culture period prolonged up to 17 d appears to be incompatible with normal oocyte morphology and maintenance of the GY-stage as well. Out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at GY-stage. Several other studies have shown that small secondary bovine follicles do not grow in culture for more than 6 or 7 d (8,16,20,21). Results presented by Wandji et al. (20) showed that the somatic component of the bovine preantral follicle could grow and produce steroids despite the high incidence of oocyte death. Therefore, in the bovine ovary, the follicle would develop without maintenance of the communication between oocyte and surrounding granulosa cells. Recently, Harada et al. (9) reported that the in vitro culture of bovine oocytes dissected from bigger follicles, i.e, 500 to 700 urn in diameter, supported oocyte growth and some of these oocytes acquired maturational competence after lId (14). In sheep, Newton et al. (15) found that follicles measuring 190-240 urn in diameter survived in vitro up to 30 d and at the end of the culture period, the enclosed oocytes had grown to almost full size. Recently, ltoh et al. (10) showed that long-term survival of small bovine preantral follicles and their oocytes can be improved by co-culture with various types of somatic cells. These results supported our
259
Theriogen%gy
observation that bigger preantral and early antral bovine follicles seem to be the most promising material for in vitro culture . Maintenance of oocyte quality, characterized by preservat ion of the GV-configuration during culture periods up to 14 d, and an increase of oocyte diameter indicate that bovine oocytes in late preantral and early antral follicles can be maintained in meiotic arrest in our culture system. Further studies are needed to show the competen ce of these oocytes for maturation and fertilization in vitro Recently, Harada et al. (9) showed that supplementation of medium with 4 mM of hypoxanthine for culture of bovine antral follicles preserved the normal morphology of the follicles, and increased the number of cumulus-enclosed oocytes and percentage of ooeytes in the GV-stage. Moreover, it has been reported in cattle that the number of morphologically normal oocytes increases when preantral follicles are cultured in hypoxathine supplemented medium (6). The physiological role of hypoxanthine with respect to bovine oocytes is unclear because bovine follicular fluid does not contain levels of hypoxanthine comparable to that in other mammals (4). It has been suggested that the levels of cAMP in granulosa cells affect both their development and function (3). Hypoxanthine in the culture medium is thought to maintain cAMP levels in granulosa cells by its inhibitory action on cAMP-phosphodiesterase. It seems possible that hypoxanthine has a role in maintaining an association between the oocyte and surrounding granulosa cells of in vitro grown bovine oocytes and that the cells exert an inhibitory action on resumption of meiosis in the oocytes through this association (9). In our experiment, the culture medium was supplemented with 2 mM of hypoxanthine and there was no increase in the proportion of oocytes resuming meiosis until the culture period reached 17 days. Most of the follicles that had been cultured up to 14 d contained morphologicall y normal oocytes (i.e, germinal vesicle oocyte with compact cumulus layers) comparable with freshly isolated oocytes. The objective of obtain ing mature oocytes from late preantral and early antral bovine follicles seems now more realistic, but still requires further fundamental research. Because of the high rate of oocytes, both "fresh" and in vitro-cultured, having signs of degeneration we conclude that strict selection of follicles used for the in vitro culture should be done. However , because the oocyte in late preantral or early antral bovine follicle is only occasionall y c1carly visible under the inverted microscope such selection may be difficult. REFERENCES I. Braw-Tal R, Yossefi S. Studies in vivo and in vitro on the initiation of follicle growth in the bovine ovary. J Reprod Ferti11997; I09: 165-171. 2. Cortvindt R, Smitz J, Van Steirteghem A. In vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system. Human Reprod 1996;II: 2656-2666. 3. Eppig JJ. Mammalian oocyte development in vivo and in vitro . In: Wassarman PM (ed), Elements of Mammalian Fertilization. CRC Press. Boca Raton FL, 1991;57-76. 4. Eppig JJ, Downs SM. The role of purines in the maintenance of meiotic arrest in mammalian oocytes . In: Haseltine FP and First NL (ed), Meiotic Inhibition: Molecular Control of Meiosis . New York Alain R Liss Inc., 1988;I03-113. 5. Fair T, Hyttel P, Greve T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity . Mol Reprod Dev 1995; 42: 437-442 .
260
Theriogenology
6. Figueiredo JR, Hulshof SCJ, van den Hurk R, Nusgens B, Bevers MM, Ectors FJ, Beckers JF. Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro. Theriogenology 1994; 41: 1333-1346. 7. Gougeon A, Testart J. Germinal vesicle breakdown in oocytes of human atretic follicles during the menstrual cycle. J Reprod Fertil 1986;78: 389-401. 8. Hulshof SCl Bovine preantral follicles and their development in vitro. PhD thesis, University of Utrecht, 1995. 9. 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: 743-755. 10. Itoh T, Abe H, Yamashita S, Sendai Y, Satoh T, Hoshi H. Bovine mesenchymal-derived cells support long-term survival and growth of bovine preantral follicles in vitro. Theriogenology 1999; 51: 302 abstr. II. Katska L, Rynska B. The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes. Theriogenology 1998; 50: 213-222. 12. Lussier JP, Matton P, Dufour Jl Growth rates of follicles in the ovary of the cow. J Reprod Fertil1987; 81: 301-307. 13. McGaughey RW, Montgomery DH, Richter JD. Germinal vesicle configurations and patterns of polypeptide synthesis of porcine oocytes from antral follicles of different size, as related to their competency of spontaneous maturation. J Exp Zoology 1979; 209:239-254. 14. Miyano T, Ozaki S, Matsumura K, Miyake M, Kato S, Yamamoto K. In vitro growth, fertilisation and development of bovine oocytes from early antral follicles. Proc Serono Symp, Gametes: Development and Function. Lauria A, Gandolfi F, Enne G, Gianaroli L (ed), 1998; 575. 15. Newton H, Picton H, Gosden RG. In vitro growth of preantral follicles isolated from cryopreserved ovine tissue.. Lauria A, Gandolfi F, Enne G, Gianaroli L (eds), Proc Serono Symp Gametes: Development and Function. 1998;584. 16. Nuttinck F, Collette L, Massip A, Dessy F. Histologic and autoradiographic study of the in vitro effects of FGF-2 and FSH on isolated bovine preantral follicles: preliminary investigation. Theriogenology 1996;45: 1235-1245. 17. Schotanus K, Hage WJ, Spek ER, Bevers MM, van der Hurk R. Viability aspects of isolated bovine preantral follicles. Proc 13th ICAR, 1996; P7-27 abstr. 18. Telfer EE. The development of methods for isolation and culture of preantral follicles from bovine and porcine ovaries. Theriogenology 1996; 45: 111-120. 19. van den Hurk R, Spek ER, Hage WJ, Fair T, Ralph lH, Schotanus K. Ultrastructure and viability of isolated bovine preantral follicles. Hum Reprod Update 1998; 4: 833-841. 20. Wandji S.A., Eppig JJ, Fortune lE. FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles. Theriogenology 1996; 45: 817-832. 21. Wandji SA, Srsen V, Voss AK, Eppig JJ, Fortune JE. Initiation in vitro of growth of bovine primordial follicles. Bioi Reprod I996b; 55: 942-948.
NUCLEAR CONFIGURATION OF BOVINE OOCYTES DERIVED FROM FRESH AND IN VITRO-CULTURED PREANTRAL AND EARLY ANTRAL OVARIAN FOLLICLES L. Klitska,la H. Alm 2 and B. Rynska! IDepartment of Animal Reproduction, National Research Institute of Animal Production, Balice/Krakow, Poland; 2Department of Reproductive Biology, Research Institute for the Biology of Farm Animals, Dummerstorf/Rostock, Germany Received for publication: August 26, 1999 Accepted: May 18, 2000 ABSTRACT The aim of this experiment was to characterize the growth and nuclear configuration of oocytes isolated from late preantral and early antral bovine ovarian follicles immediately after recovery and after the in vitro culture . Individual follicles were isolated by microdissection from slices of the ovarian cortex . Follicles were sorted by diameter into 175 to 224, 225 to 274 and 275 to 325 urn-size classes. The follicles selected for in vitro culture were placed singly into 40 ul. droplets of medium (TCM 199 enriched with FCS, insulin, transferrin , sodium selenite, sodium pyruvate , l-glutarnine, hypoxanthine , FSH and estradiol-Up) and cultured for 6, 8, II, 14 or 17 d. The sizes of follicles and oocytes were related to the duration of culture and gradually increased as culture duration was prolonged. The analysis of the relationship between mean diameters of oocytes at the time of recovery and after the in vitro culture, has shown significant differences after culture lasting 8 d (76.9±9.9 vs. 86.1±1l.lllm; P< 0.05), II d (77.0±9.9 vs. 91.9±17.5 11m; P< 0.01), 14 d (80.0±9.5 vs. 97.9±16.5 11m; P< 0.01) and 17 d (82.6±6.6 vs. 97.2±11.5 11m; P< 0.01). No statistical differences were shown among oocytes in the 5 pre-culture groups (79.5±8.8 ; 76.9±9.9; 77.1±9.9; 80.l±9.5 and 82.6±6.6 urn). Meiotic arrest was preserved in 71.9 % of oocytes in our culture system up to 14 d. Frequency of the germinal vesicle (GV) stage did not significantly differ among oocytes evaluated "fresh" or cultured for 6, 8, II or 14 d. No relationship was observed between the size class of follicles and the frequency of the GV-stage. Prolonging the culture period to 17 d drastically decreased the percentage of oocytes in the GVstage (18.7 %) and increased the percentage of oocytes having premature initiation of meiosis (GVBD; 46.3 %) and degeneration (25.0 %). These results suggest that out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at the GV-stage. @
2000 by Elsevier Science Inc.
Key words: cattle, preantral follicle, in vitro culture, oocyte, germinal vesicle
Acknowledgments The authors thank Dr. K. Hinrichs of the Texas A&M University for linguistic review and critical reading of the manucript, Dr. R. Poehland for the computer processing of the pictures , and M.S. H. Korzonek for statistical help. a Correspondence and reprint requests: Lucyna Katska, National Research Institute of Animal Production, Department of Animal Reproduction , 32-083 Balice/Krakow, Poland. Tel. (48)( 12) 285 6714; e-mail : [email protected] Theriogenology 54:247-260, 2000 Cl2000 Elsevier SCience Inc.
0093·691X!OO/$-see front matter PII: SOO93·691X(OOlOO345-9
248
Theriogenology INTRODUCTION
In cattle, a follicle that has formed an antrum needs 40 d to reach ovulatory size (12). By extrapolation of these data it was estimated that the preantral stage (primary or secondary) follicle requires at least 60 to 80 d to reach maturity. Small preantral follicles (diameter 35 to 100 urn) can survive in culture up to 6 or 7 d, and during this time cell proliferation and follicle growth are observed (1,6,8,17,20,21). However, the quality of oocyte ultrastructure appears to be unsatisfactory not only after culture but even immediately after follicle isolation. Recently, Schotanus et al. (17) and van den Hurk et al. (19) have reported that over 90% bovine preantral follicles (less than 60 urn in diameter), isolated mechanically by a tissue chopper, showed a poor uItrastucture, especially in their oocytes. These follicles were characterized by swollen mitochondria, a wrinkled nuclear membrane, and vacuoles in the cytoplasm of the oocyte or in the cytoplasm of both the granulosa cells and the oocyte. Morphology was considered normal when it resembled that of the follicles in the ovarian slices (17). In contrast with the small follicles, the percentage of ultrastructurally poor large preantral follicles had decreased to 27 % after 5 d of culture, possibly due to betterisolation and culture conditions (19). Recently, Harada et al. (9) reported that bovine oocytes isolated from early antral follicles 0.5-0.7 mm in diameter successfully grew during in vitro culture for 11 d and their diameter increased from 90-99 urn to 108.0 ± 5.3 11m. Some oocytes from follicles of this size, similarly cultured as in the Harada et a1. (9) experiment, were able to reach meiotic competence, fertilization and developmental competence as well (14). We have reported in a previous experiment (II), that the survival time of follicles is related to their initial size at the beginning of culture. The longest period of growth was observed for follicles 275 to 324 um in diameter (i.e, 10.7±5.7; 12.1±6.2 and 12.2±2.7 d, respectively, for culture in supplemented' Menezo B2, TCM 199 enriched with fetal calf serum (FCS), and TCM 199 enriched with steer serum. Survival and growth of some follicles were maintained for 23 d. Moreover, we have shown the positive influence of FSH, estradiol-17 13, hypoxathine, insulin, transferrin, sodium selenite, sodium pyruvate, and l-glutamine on follicle survival and growth of medium supplementation (11). There are now reports on several culture systems that support bovine preantral follicle development for a few days (reviewed by Telfer (18) and van den Hurk et al. (19) but these studies present little information on nuclear configuration of oocytes, especially when they are recovered from late preantral and early antral follicles. During the prolonged culture of follicles, it is essential to know whether the follicle maintains meiotic arrest of the oocyte and how the oocyte survives in the culture. The preliminary results of our previous study (II) have indicated that the morphologically normal structure of oocytes (i.e. germinal vesicle oocyte with compact, dense-cumulus layers and homogenous, intact cytoplasm) was preserved only in approximately 35% of the follicles that survived in vitro culture for more than 2 wk. It has seemed to us, therefore, essential to characterize nuclear configuration of oocytes both immediately after recovery of follicle and after in vitro culture, using late preantral and early antral follicles.
249
Theriogenology MATERIALS AND METHODS
The methods for follicle recovery and culture were based on those described in our previous experiment (11) . Briefly, ovaries were collected from heifers and cows at a local abattoir and were transported to the laboratory within I h. The ovaries were washed 4 times in PBS supplemented with kanarnycins (0.075 gIL), and thin cortical slices « 1.0 mm ofthiekness) were prepared with a razor blade. The slices were stored in a HEPES-buffered medium TCM 199 containing 5% Fcsa and antibiotics, at room temperature. Individual preantral and very early antral follicles were isolated by microdissection of the ovarian slices (teased from adhering stroma) using fine sharp needles (30 g 1/2 inch) fitted to 1mLsyringe barrels or acupuncture needlesb (0.25 diameter) under a stereomicroscope at x 80 magnification. Follicle isolation was carried out over approximately 2 h of manipulation (II). Intact preantral and early antral follicles were sorted by diameter as previously described (II) into 175 to 224, 225 to 274 and 275 to 325 um size classes. The follicles surrounded by an intact basal membrane and possessing clear and compact granulosa cell layers were selected for the experiment. Approximately one-third of selected follicles served as a control; they were ruptured immediately after recovery and isolated oocytes were measured by diameter and fixed for evaluation of nuclear configuration. The follicles used for in vitro culture were randomly distributed across the different times of cultu re. Follicles were individually transferred into droplets of medium in a 60-mm Petri dish C conta ining 14 x 40 ul, droplets, covered with 5 mLof the mineral oil a . On day 2 of the culture, the medium was replenished by removing 20 ~L and replacing it with 20 ul, of fresh medium , and thereafter half of the medium was refreshed every 48 h. The culture medium was composed of TCM 199 with 10% heat-inactivated FCS, ITS (insulin 6.25 ug/rnl., transferrin 6.25 ug/ml, and sodium selenite 6.25 ng/rnl.)«, 0.23 mM sodium pyruvates, 2 mM Lsglutaminee, 2 mM hypoxanthines, FSHa (from human pituitary, 0.05 IU/mL) and estradiol-l Zp'' (l)lg/ mL) (II). Follicles were cultured in an incubator at 38.5°C, 100% humidity and 5% C02 in air, for 6, 8, II, 14 or 17 d. Characterization of the cultured follicles was done on Day 0 by means of a phase-contrast inverted microscope. Follicle growth was monitored daily by measurement of follicular diameter using a precalihrated ocular micrometer on an inverted microscope at x 100 magnification. Follicular survival in culture was considered positive as long as the basal membrane remained intact and no visible signs of degeneration were noticed. Morphological criteria of follicular degeneration after culture included the following signs : follicular outline becoming irregular and dark patches of dead cells appearing within the membrana granulosa, especially around the oocyte . Oocytes were freed from their follicles immediately after recovery ("fresh") and after 6, 8, II, 14 or 17 d of the in vitro culture. Oocyte size assessment was carried out after mechanical removal of the oocytes from their follicles under a stereomicroscope. Follicles were punctured with thin , sharp acupuncture needles until oocytes were released into the manipulation medium . Those oocytes that still showed the presence of follicle cells surrounding the zona pellucid" were gently pipettcd through a narrow glass pipette. Only oocytes free of follicle cells were used for further evaluation. Oocyte quality was recorded immediately at the time of follicle recovery
Sigma Chemical Company, S1. Louis, MO , USA. b Acumedic LTD, London, UK. C Falcon, Becton, Dickinson, USA.
a
Theriogen%gy
250
("fresh") and after 6, 8, II, 14 or 17 d of culture. The evaluation of oocyte quality included the size of the oocyte with or without the zona pellucida and nucleus configuration. Nuclear and chromatin status was evaluated after oocyte had been mounted onto a slide, fixed in acetic ethanol (CH3COOH: C2H50H= I :3), stained with I% aceto-orcein and examined under phasecontrast microscope at x 1000 magnification. Statistical differences in the diameters of the oocyte were analyzed by the Student's t-test. Proportion of oocytes in different stage of meiosis was compared using the Chi-square test. Differences with P<0.05 were considered to be statistically significant. RESULTS A total of 392 bovine follicles with a mean diameter of approximately 250 urn were analyzed. Figure I shows an example of a follicle at the time of recovery that was used for the experiment. During culture, cells originating from the surface of the follicle attached themselves to the dish and proliferated, forming a monolayer surrounding the follicle and attaching it to the dish (Figure 2). By Day II, almost all follicles had become attached to the dish and the follicles reached a "spread appearance" (Figure 3). The size of the antral cavity (indicated by the appearance of gaps within the granulosa cell mass) gradually increased (Figures 3 and 4). More than 50 % of the oocytes freed from their follicles maintained oocyte-granulosa connections (Figure 5). Other oocytes lost connections with the granulosa cells (denuded oocytes), degenerated or completely disappeared while granulosa cells appeared healthy. Denuded oocytes were saved for further evaluation while oocytes with heavy degenerated cytoplasm were discarded. Table I. Classification and characterization of bovine oocytes preantral and early antral ovarian follicles
isolated from
fresh, late
Follicle size classes
Evaluated follicles
Follicle diameter, f.!m
No. (%) of recovered
(urn)
(n)
(mean±SO)
oocytes
(mean ± SO) with zpa without zpa
175-224
14
198.3 ± 14.4
6 (42.9)
85.6 ± 9.5
70.5 ± 3.5
225-274
30
248.7 ± 13.1
21 (70.0)
79.5 ± 8.8
64.0 ± 8.5
304.1 ± 16.2
10 (83.3)
85.9 ± 8.9
69.2 ± 6.3
275-324 12 azp - the zona pellucida
Oocyte diameter, urn
The relationships between follicle diameter before and after culture and oocyte diameter with and without zona pellucida are shown in Tables I and 2. Diameter of both follicles and oocytes was related to the culture period and gradually increased as the culture time was prolonged. The analysis of the relation between mean diameters of the oocyte at the time of recovery and after in vitro culture has shown significant differences after culture lasting 8 d (76.9±9.9 vs. 86.1± 11.1 urn; P< 0.05), II d (77.1±9.8 vs. 91.9± 17.5 urn; p< 0.01), 14 d (80.0±9.5
251
Theriogenology
Figure I. Freshly isolated bovine earlyantral follicle. Scale bar represents 50 /.lID. 00: oocyte; arrow: basal lamina.
-Figure 2. Follicle attached to the culture dish after 8 days of in vitro culture . Scale bar represents 100 ).lID. 00: oocyte; arrow: basal lamina .
252
Theriogenology
Figure3. Follicle reaching "spreadappearance" after 11daysof in vitroculture. Antrum visible as emptypatches withinthe granulosa cell mass. Scalebar represents 100IJ.m. 00: oocyte; A: antrum; arrow: basallamina.
Figure 4. The samefollicle as on Figure 3 but after 17daysof in vitroculture. Increase of antrumclearlyvisible. Scalebar represents 100IJ.m. 00: oocyte; A: antrum.
253
Theriogenology
/
I
Figure 5. Oocyte freed from the follicle after I I days of in vitro culture. Oocyte surrounded by compact and dense cumulus cell layers. Scale bar represents I00 urn, 00: oocyte.
Figure 6. Oocyte in GV stage after 14 days ofin vitro culture. Scale bar represents germinal vesicle; arrow: nucleolus.
1O~.
GV:
I\)
~
Table 2. Characterization of bovine oocytes derived from fresh and in vitro-cultured late preantral and early antral ovarian follicles
Duration Follicles of culture totala/cult. (d) N
Diameters before culture, 11m, mean ± SO, (n) Follicle
Oocyte with Zpb
Oocyte without Zpb
Diameters after culture, 11m, mean ± SD, (n) Follicle
Oocyte with
zr"
Oocyte without Zpb
6
88/58
245.9 ± 54.6
c 76.6 ± 8.8 (28)
c 64.0 ± 8.5 (28)
330.1 ± 76.6
c 79.5 ± 8.8 (30)
8
58/45
244.7 ± 42.7
c 76.9 ± 9.9 (13)
c 64.0 ± 8.5 (13)
373.0 ± 9804
86.1 ± IUd (33)
c 69.5 ± IlA (33)
II
43/22
253.7 ±42.9
c 77.1 ± 9.9 (19)
c 64.0 ± 8.5 (19)
393.4 ± 1l1.8
e 91.9 ± 17.5 (19)
67.9 ± 16f (19)
14
92/35
253.7 ± 40.0
c 80.0 ± 9.5 (52)
c 66.6 ± 7.5 (52)
456.9 ± 114.2
e 97.9 ± 16.5 (35)
d 80.3 ± 21.2 (35)
17
55/39
265.5 ± 40.3
c 82.6 ± 6.6 (14)
c 69.2 ± 6.3 (14)
654.6 ± 172.9
97.2 ±
n.s" (14)
80.3 ± llA (14)
~otal
69.8 ± 9 f (30)
c
no. Include the control (fresh) and the in vitro cultured follicles used for measurement of the initial diameter.
bZp _ zona pellucida.
cod. c:eComparisons were made within the row between diameters of fresh and cultured oocytes, means with different superscripts are significantly different (d p<0.05, c:ep
:;t m
5'
~o
c-
~
Theriogenology
255
vs. 97.9±16.5 um; P< 0.01) and 17 d (82.6±6.6 vs. 97.2±11.5 urn; P< 0.01). No statistical differences were shown in the oocyte diameters among the 5 pre-culture groups (Table 2). Meiotic arrest was maintained in oocytes up to 14 d of in vitro culture. The frequency of the germinal vesicle (OV) stage did not differ significantly between oocytes evaluated as "fresh" and those cultured for 6,8, II and 14 d, being 75.7; 59.5; 60.0; 75.1 and 71.9 %, respectively (Table 3, Figure 6). No relationship was observed between follicle size and frequency of the OV-stage in fresh oocytes and those cultured up to 14 d (Figures 7a to e). However, prolonging the culture period' to 17 d decreased the percentage of oocytes in GV-stage (18.7 %), and increased the incidence of premature meiosis initiation and oocyte degeneration (Table 3 and Figure 7f). A lower percentage of oocytes (41.0 %) was recovered after the culture was prolonged up to 17 d in comparison to those released from the follicles and cultured for 6 to 14 d. Approximately 43 to 83% of the oocytes recovered from uncultured follicles (Table I) and 41 to 91 % of the oocytes from the in vitro cultured follicles (Table 3) were analyzable. The rest of the oocytes that were not used for the microscopic investigation, included those not recovered, absent or lost during manipulations, i.e, broken on isolation, lost during fixation/staining, very tightly covered by cumulus cells' (nuclear configuration undetermined) and/or with completely degenerated cytoplasm t'empty" zona pellucida). DISCUSSION In cattle, oocytes in early antral follicles do not reach their final size of 120 urn until after the antrum has formed (5). The growth period appears to be considerably long. Therefore, a culture system supporting growth of both follicle and oocyte should be developed. Culture efficacy can be characterized by maintenance of a germinal vesicle configuration and a normal oocyte morphology through the period of growth. Defined homogenous oocyte population in GVstage at recovery is essential for effectiveness of the IYMlIVF technique. In our previous experiment (II) on isolation and in vitro culture of late preantral and early antral bovine ovarian follicles, we reported that the total growth, growth rate and survival of follicles of different size were similar in the 2 media (TCM 199 and Menezo B2). However, the culture of follicles in the TCM 199 that was supplemented only with steer serum significantly reduced follicular survival and growth in comparison with follicles cultured in the medium containing other additives such as FSH, ITS, I-glutamine, sodium pyruvate and hypoxanthine. Some late preantral and early antral follicles survived and grew up to 23 d, but their mean growth span did not exceed 10 to 12 d. Even though considerable differences were noticed in the length of time follicles survived in vitro, there were no obvious differences in the follicular growth rate per day of culture and size increase after culture. The highest growth rates of follicles were observed at the beginning of culture and a gradual reduction of these rates during the culture period was noted. The preliminary analysis of nuclear configuration in oocytes freed from follicles that had survived in vitro culture for more than 2 wk has indicated that GY-configuration was preserved only in approximately 35% of the oocytes (II). Therefore, it has seemed to us essential to determine the frequency of GY-configuration in oocytes freed from follicles immediately after their recovery and after several days of the in vitro culture and to establish the relationship between the size of follicle and/or the duration of the in vitro culture and the preservation of OV-configuration.
I\:)
~ (a)
ro)
Control (fresh ooc ytes)
>
*'
l;l, 60.0
60.0 60.0
~ 0
40.0 ' -
~
20.0
~
20.0 · -
-
QO
0.0 175224
125174
225274
275324
>325
d
.
175224
(e)
_I
100.0
I
60.0
II ~
-
-
---
60.0
--
40.0
-
20.0
-
-
-
-
-
-
-
-
225274
275324
follicularsize classes
-
~
40.0
-
;.
20.0 ·
-
-
QO .
.
225274
275324
>325
I
-
~
>325
I
(f)
~
-- -
> ~0
1-
40.0 20.0
.
175224
~
1-
225274
275324
>325
folliculars ize class es
--
-
.
175224
275324
>325
-
.
225274
Mean
17 days ofcuhure 1 00 ~
125174
Mean
-
foll icular size classes
60.0 l;l, ~ 60.0
> ~0
-
-
-
.
125174
Mean
14 days ofcuhure
0.0 175224
-
60.0 60.0
100.0
0.0 125174
-
;
follicular size classe s
I I days of culture
)-
-
.
125174
Mean
-
foll icular sill: classe s
r
8 days ofculture
'OO'1iifij 'OO'iiim ~ ::~ - ==== - -
100.0
~
(c)
6 days ofcuhure
60 0 60 0 ~O
mo QO
Mean
-~l
125174
175224
225274
275324
>325
_
Mean
follicularsize classes
Figure 7. Percentage of oocytes in GV stage. Oocytes isolated from fresh (a) and in vitro (b to f) bovine preantral and early antral follicles. (The initial diameter of follicles was used for classification.)
~
Q)
g.
1 is'
~
:;! (1)
g. Tabl e 3. Nuclear status of oocytes recovered from fresh and in vitro-cultured preantral and early antral bovine ovarian follicles
~o
0-
~ Culture time
Follicles
(d)
(n)
Oocytes recovered
Germinal vesicle
n (%)
Stage of meiosis, No. ( %) of oocytes Germinal vesicle breakdown
Degenerated
a Intact 4 (10.8)
b Atretic 3 (8.1)
c 2 (5.4)
0
56
37(66.1)
28 (75.7)
Total c(IS .9) 7
6
55
37 (67.3)
c 22 (59.5)
SC (2 1.6)
2 (6.4)
6 (16.2)
c 7 (18.9)
8
45
30 (66.7)
18c (60.0)
7c (23.3)
3 (10.0)
4 (13.3)
c 5 (16.7 )
11
22
16 (72.7)
12c (75.1)
3c (18.S)
I (6.3)
2 (12.5)
IC(6.3)
14
35
32 (91.4)
23 c (71.9)
6c (18.8)
4 (12.5)
2 (6.3)
c 3 (9.4 )
17
39
16 (41.0)
3e (18.7)
ge (46.3)
6 (37.5)
3 (IS .S)
d 4 (25.0)
c
a Intact GVBD- oocyte with diakinesis chromat in, the nuclear envelope partially or completely dispersed. b
Atretic GVBD- oocyte with highly condensed, pyknotic chromatin, nuclear envelope partially or completely dispersed .
cod; c:eComparisons were made within the columns ; values with different superscripts are significantly different (c:dp <0.05, c:cp< O.OI, X2 test). I\)
~
258
Theriogenology
The results of the present study showed that a relatively high proportion of fresh late preantral and early antral bovine follicles contained oocytes that appear to have undergone resumption of meiosis. Because of the very small follicle size, this high percentage in GYBD probably represents atresia. Resumption of meiosis may occur spontaneously in atretic ovarian follicles (7,13). Our results demonstrate that late preantral and early antral follicles with a mean diameter of approximately 250 flm increased in size during culture period lasting up to 17 d. As we have already mentioned, during the culture spindle-shaped cells, which probably represented fibroblasts originating from surface of the follicle (the rest of ovarian stroma cells and/or the theca cells), attached themselves to the dish and proliferated, with the formation of a monolayer surrounding and attaching the follicle to the dish. The initial follicular structure was lost and follicles developed a "diffuse appearance". A similar phenomenon was reported by Cortvrindt et al. (2) for in vitro-cultured mouse preantral follicles, but the loss of normal three-dimensional follicular structure did not affect oocyte quality and function of all components, i.e. theca, granulosa and oocyte. In our experiment follicular growth was accompanied by growth of oocytes freed from these follicles after 8 to 17 d of the in vitro culture. A higher rate of oocyte growth comparing to values for fresh, uncultured oocytes was observed after culture lasting longer than 8 d. The oocyte growth was noted also after 17 d of culture, however the quality of oocytes was unsatisfactory. However, it should be pointed out that the oocyte in late preantral or early antral bovine follicle is only occasionally clearly visible under the inverted microscope, therefore measurements of the oocyte diameter before and after culture could not be carried out. For this reason, the calculation of oocyte growth was done by the comparison of mean diameter of the oocytes recovered from the same size class of follicles that were freshly isolated or cultured in vitro. As we have already mentioned, the final arbiter of successful follicle growth in vitro is the quality of the oocyte. Although follicles cultured for 17 d grew, when they were mechanically dissected, most of the oocytes recovered were degenerated or were no longer in meiotic arrest. Morphological analysis of these follicles revealed signs of degeneration. In these follicles, disruption of intercellular contacts or build up of metabolic degradation products may have resulted in loss of the follicle's ability to maintain meiotic arrest, resulting in a decreased rate of the Gv-stage, premature resumption of meiosis in the oocytes and high incidence of oocyte degeneration. Therefore, the culture period prolonged up to 17 d appears to be incompatible with normal oocyte morphology and maintenance of the GY-stage as well. Out of all culture periods used in our experiment, Day 14 was found to be the longest culture time allowing for both oocyte growth and maintenance of nuclear configuration at GY-stage. Several other studies have shown that small secondary bovine follicles do not grow in culture for more than 6 or 7 d (8,16,20,21). Results presented by Wandji et al. (20) showed that the somatic component of the bovine preantral follicle could grow and produce steroids despite the high incidence of oocyte death. Therefore, in the bovine ovary, the follicle would develop without maintenance of the communication between oocyte and surrounding granulosa cells. Recently, Harada et al. (9) reported that the in vitro culture of bovine oocytes dissected from bigger follicles, i.e, 500 to 700 urn in diameter, supported oocyte growth and some of these oocytes acquired maturational competence after lId (14). In sheep, Newton et al. (15) found that follicles measuring 190-240 urn in diameter survived in vitro up to 30 d and at the end of the culture period, the enclosed oocytes had grown to almost full size. Recently, ltoh et al. (10) showed that long-term survival of small bovine preantral follicles and their oocytes can be improved by co-culture with various types of somatic cells. These results supported our
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observation that bigger preantral and early antral bovine follicles seem to be the most promising material for in vitro culture . Maintenance of oocyte quality, characterized by preservat ion of the GV-configuration during culture periods up to 14 d, and an increase of oocyte diameter indicate that bovine oocytes in late preantral and early antral follicles can be maintained in meiotic arrest in our culture system. Further studies are needed to show the competen ce of these oocytes for maturation and fertilization in vitro Recently, Harada et al. (9) showed that supplementation of medium with 4 mM of hypoxanthine for culture of bovine antral follicles preserved the normal morphology of the follicles, and increased the number of cumulus-enclosed oocytes and percentage of ooeytes in the GV-stage. Moreover, it has been reported in cattle that the number of morphologically normal oocytes increases when preantral follicles are cultured in hypoxathine supplemented medium (6). The physiological role of hypoxanthine with respect to bovine oocytes is unclear because bovine follicular fluid does not contain levels of hypoxanthine comparable to that in other mammals (4). It has been suggested that the levels of cAMP in granulosa cells affect both their development and function (3). Hypoxanthine in the culture medium is thought to maintain cAMP levels in granulosa cells by its inhibitory action on cAMP-phosphodiesterase. It seems possible that hypoxanthine has a role in maintaining an association between the oocyte and surrounding granulosa cells of in vitro grown bovine oocytes and that the cells exert an inhibitory action on resumption of meiosis in the oocytes through this association (9). In our experiment, the culture medium was supplemented with 2 mM of hypoxanthine and there was no increase in the proportion of oocytes resuming meiosis until the culture period reached 17 days. Most of the follicles that had been cultured up to 14 d contained morphologicall y normal oocytes (i.e, germinal vesicle oocyte with compact cumulus layers) comparable with freshly isolated oocytes. The objective of obtain ing mature oocytes from late preantral and early antral bovine follicles seems now more realistic, but still requires further fundamental research. Because of the high rate of oocytes, both "fresh" and in vitro-cultured, having signs of degeneration we conclude that strict selection of follicles used for the in vitro culture should be done. However , because the oocyte in late preantral or early antral bovine follicle is only occasionall y c1carly visible under the inverted microscope such selection may be difficult. REFERENCES I. Braw-Tal R, Yossefi S. Studies in vivo and in vitro on the initiation of follicle growth in the bovine ovary. J Reprod Ferti11997; I09: 165-171. 2. Cortvindt R, Smitz J, Van Steirteghem A. In vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system. Human Reprod 1996;II: 2656-2666. 3. Eppig JJ. Mammalian oocyte development in vivo and in vitro . In: Wassarman PM (ed), Elements of Mammalian Fertilization. CRC Press. Boca Raton FL, 1991;57-76. 4. Eppig JJ, Downs SM. The role of purines in the maintenance of meiotic arrest in mammalian oocytes . In: Haseltine FP and First NL (ed), Meiotic Inhibition: Molecular Control of Meiosis . New York Alain R Liss Inc., 1988;I03-113. 5. Fair T, Hyttel P, Greve T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity . Mol Reprod Dev 1995; 42: 437-442 .
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6. Figueiredo JR, Hulshof SCJ, van den Hurk R, Nusgens B, Bevers MM, Ectors FJ, Beckers JF. Preservation of oocyte and granulosa cell morphology in bovine preantral follicles cultured in vitro. Theriogenology 1994; 41: 1333-1346. 7. Gougeon A, Testart J. Germinal vesicle breakdown in oocytes of human atretic follicles during the menstrual cycle. J Reprod Fertil 1986;78: 389-401. 8. Hulshof SCl Bovine preantral follicles and their development in vitro. PhD thesis, University of Utrecht, 1995. 9. 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: 743-755. 10. Itoh T, Abe H, Yamashita S, Sendai Y, Satoh T, Hoshi H. Bovine mesenchymal-derived cells support long-term survival and growth of bovine preantral follicles in vitro. Theriogenology 1999; 51: 302 abstr. II. Katska L, Rynska B. The isolation and in vitro culture of bovine preantral and early antral follicles of different size classes. Theriogenology 1998; 50: 213-222. 12. Lussier JP, Matton P, Dufour Jl Growth rates of follicles in the ovary of the cow. J Reprod Fertil1987; 81: 301-307. 13. McGaughey RW, Montgomery DH, Richter JD. Germinal vesicle configurations and patterns of polypeptide synthesis of porcine oocytes from antral follicles of different size, as related to their competency of spontaneous maturation. J Exp Zoology 1979; 209:239-254. 14. Miyano T, Ozaki S, Matsumura K, Miyake M, Kato S, Yamamoto K. In vitro growth, fertilisation and development of bovine oocytes from early antral follicles. Proc Serono Symp, Gametes: Development and Function. Lauria A, Gandolfi F, Enne G, Gianaroli L (ed), 1998; 575. 15. Newton H, Picton H, Gosden RG. In vitro growth of preantral follicles isolated from cryopreserved ovine tissue.. Lauria A, Gandolfi F, Enne G, Gianaroli L (eds), Proc Serono Symp Gametes: Development and Function. 1998;584. 16. Nuttinck F, Collette L, Massip A, Dessy F. Histologic and autoradiographic study of the in vitro effects of FGF-2 and FSH on isolated bovine preantral follicles: preliminary investigation. Theriogenology 1996;45: 1235-1245. 17. Schotanus K, Hage WJ, Spek ER, Bevers MM, van der Hurk R. Viability aspects of isolated bovine preantral follicles. Proc 13th ICAR, 1996; P7-27 abstr. 18. Telfer EE. The development of methods for isolation and culture of preantral follicles from bovine and porcine ovaries. Theriogenology 1996; 45: 111-120. 19. van den Hurk R, Spek ER, Hage WJ, Fair T, Ralph lH, Schotanus K. Ultrastructure and viability of isolated bovine preantral follicles. Hum Reprod Update 1998; 4: 833-841. 20. Wandji S.A., Eppig JJ, Fortune lE. FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles. Theriogenology 1996; 45: 817-832. 21. Wandji SA, Srsen V, Voss AK, Eppig JJ, Fortune JE. Initiation in vitro of growth of bovine primordial follicles. Bioi Reprod I996b; 55: 942-948.