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Effect of hormones and growth factors on in vitro development of sheep preantral follicles
Small Ruminant Research 70 (2007) 93–100
Effect of hormones and growth factors on in vitro development of sheep preantral follicles G. Arunakumari a , R. Vagdevi a , B.S. Rao a,1 , B.R. Naik a , K.S. Naidu a,2 , R.V. Suresh Kumar a,b , V.H. Rao a,∗ a
Embryo Biotechnology Laboratory, Department of Physiology, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati 517502, India b Department of Surgery and Radiology, India
Received 19 January 2005; received in revised form 4 January 2006; accepted 4 January 2006 Available online 3 April 2006
Abstract The present investigation attempts to improve the frequency of in vitro maturation of oocytes by culturing small (150–250 m) and large (>250–400 m) preantral follicles (PFs) of sheep for 6 days in various combinations/sequences of thyroxin (T4 ), FSH, LH, transforming growth factor alpha (TGF-␣), epidermal growth factor (EGF) and heat-treated foetal calf serum (FCS). Bicarbonatebuffered tissue culture medium 199, supplemented with 50 g ml−1 gentamicin sulphate, served as the control medium. In vitro development was initially assessed by the proportion of PFs exhibiting an increase in size, mean increase in diameter and antrum formation. Nuclear maturation to the metaphase II stage of the oocytes isolated from cultured PFs, after an additional 24-h in vitro maturation, indicated success. A total of 15% of oocytes from small PFs and 55% from large PFs, cultured in T4 + FSH, matured to metaphase II. Culture of PFs in other combinations/sequences of hormones and growth factors, including the control medium, supported a significantly lower proportion of oocytes maturing to metaphase II stage. It is concluded that 6-day in vitro culture of sheep PFs in thyroxin and FSH greatly improves the frequency of oocyte maturation to metaphase II stage. © 2006 Elsevier B.V. All rights reserved. Keywords: Sheep; Preantral follicles; Thyroxin; Growth factors; FSH
1. Introduction Biotechnology tools, such as embryo transfer, embryo cryopreservation and in vitro production of embryos, have only enhanced the utilisation of female genetic
∗ Corresponding author. Tel.: +91 877 2249376; fax: +91 877 2249563. E-mail address: [email protected] (V.H. Rao). 1 Present address: Center for Cellular and Molecular Biology, Hyderabad, India. 2 Present address: Department of Animal Reproduction and Gynecology, India.
0921-4488/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.01.002
resources to a limited extent, as full advantage is not taken of the large number of potential oocytes present in primordial and preantral follicles (PFs) in the ovaries. Most of the oocytes in PFs never become available for ovulation and fertilization. Therefore, to maximize the utilization of female gametes, repeatable in vitro techniques for maturing PFs and subsequent fertilization of oocytes are desirable. Other applications of in vitro maturation of PFs have been detailed previously (Mao et al., 2002; Hemamalini et al., 2003; Demeestere et al., 2005; Tamilmani et al., 2005). Only in the mouse has in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes from PFs resulted
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G. Arunakumari et al. / Small Ruminant Research 70 (2007) 93–100
in the birth of live offspring (Nayudu and Osborn, 1992; Boland et al., 1993; Eppig and Schroeder, 1989; Carroll et al., 1990; Eppig and O’Brien, 1996; Liu et al., 2001). Follicular antrum development in cultured PFs of pig (Hirao et al., 1994) and cow (Sviridov and Kaganskaya, 1991; Nuttinck et al., 1993; Braw-Tal and Yossefi, 1997; Gutierrez et al., 2000) has also been accomplished. In the pig, embryos were produced from oocytes collected from cultured PFs (Wu et al., 2001). Chelikani et al. (1998) reported the isolation of PFs from sheep and goat ovaries for the first time. Antrum development in cultured sheep PFs has also been reported (Cecconi et al., 1999), while success in the growth and new DNA synthesis in cultured sheep and goat PFs was only recently achieved (Hemamalini et al., 2003; Rajarajan et al., 2006). Recently, 30% of oocytes from sheep PFs cultured in TGF-␣ or FSH could be matured to MII stage for the first time (Tamilmani et al., 2005). However, meiotic maturation rates in excess of 80% for oocytes isolated from the antral follicles of sheep have been reported earlier (Rao et al., 2002). The reported success in meiotic maturation of sheep oocytes from cultured PFs needs to be improved, however (Tamilmani et al., 2005). Several endocrine, paracrine and autocrine factors are involved simultaneously and/or in a specific sequence during the complex process of ovarian folliculogenesis (Demeestere et al., 2005). Although the importance of thyroxin in the growth and development of ovarian follicles is well known (Dickson, 1996), it has never been used in the culture medium for PFs in domestic animals. Thus, the goal of the present study was to improve the meiotic maturation rate of oocytes by in vitro culture of sheep PFs in different combinations/sequences of thyroxin, FSH, LH, EGF and TGF-alpha. 2. Materials and methods Unless otherwise stated, all culture media, hormones, growth factors, fetal calf serum (FCS) and chemicals were purchased from the Sigma (St. Louis, MO, USA) and plastics from Nunclon (Roskilde, Denmark). Barring the media supplemented with different growth factors and hormones all other solutions were filtered through a 0.22-m sterilizing filter (Sartorius, Germany) prior to use. All media were incubated at 39 ◦ C under a humidified atmosphere of 5% CO2 in air for 1 h prior to use.
tissue culture medium 199 (TCM199H), supplemented with 25 IU ml−1 heparin and 50 g ml−1 gentamicin sulphate], 0.5% bovine serum albumin (BSA), stock solutions of TGF-␣, EGF, FSH and LH have all been described previously (Tamilmani et al., 2005). The source, potency and endotoxin levels for the different growth factors and hormones used in this study were the same as those described by Tamilmani et al. (2005). All the stock solutions were stored at −20 ◦ C until used. Thyroxin stock solution was prepared by dissolving a 100-g thyroxin sodium tablet (IP) (ELTROXIN, Glaxo India Limited) in 10 ml bicarbonate-buffered tissue culture medium 199 (TCM199B), supplemented with 50 g ml−1 gentamicin sulphate, to yield a concentration of 10 g ml−1 . Thyroxin-supplemented culture medium was prepared by diluting 500 l of the thyroxin stock solution in 4500 l of control medium to obtain a final concentration of 1 g ml−1 thyroxin (T4 ). Supplementation of the culture medium with one or more of the other hormones and growth factors was achieved by appropriately mixing thyroxinsupplemented medium with appropriate volumes of the stock solutions. For example, supplementation of the culture medium with thyroxin 1 g ml−1 and TGF-␣ 2.5 ng ml−1 was achieved by adding 1995 l thyroxinsupplemented culture medium to 5 l of the TGF-␣ stock solution. Similarly, supplementation of the culture medium with thyroxin 1 g ml−1 , FSH 2 g ml−1 and LH 1 g ml−1 was achieved by mixing 1970 l of thyroxin-supplemented medium, 28 l FSH stock solution and 2 l LH stock solution. Various supplemented media were stored for up to 1 week at 4 ◦ C in a disposable syringe and used directly without filtering. TCM199B, supplemented with 50 g ml−1 gentamicin sulphate, was used as the control medium. This was filtered through a 0.22-m filter and stored at 4 ◦ C for up to 1 week. TCM199B, supplemented with 0.5 g ml−1 FSH, 100 g ml−1 LH, 1 g ml−1 estradiol 17, 50 g ml−1 gentamicin sulphate, 10 g ml−1 BSA and 20% (v/v) heat-treated oestrus sheep serum was used for the in vitro maturation (IVM) of oocytes collected from the cultured PFs (Rao et al., 2002). This was stored at 4 ◦ C for up to 1 week and equilibrated for 1 h at 39 ◦ C in 5% CO2 in air, prior to use. 2.2. Collection of ovaries, isolation, selection and culture of PFs
2.1. Culture media Preparation of phosphate-buffered saline (PBS), the handling medium for PFs and oocytes [HEPES-buffered
Collection of ovaries, isolation of PFs, classification into small and large sizes, selection of PFs for cultures were performed, as described by Tamilmani et al. (2005).
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All the small and large follicles isolated on any day were randomly allotted to one of the 15 treatments and each treatment was replicated seven times. Culture procedures for PFs were followed, as standardized previously (Hemamalini et al., 2003; Tamilmani et al., 2005). Briefly, the appropriate culture medium was pre-incubated for 1 h at 39 ◦ C in 5% CO2 in air in a CO2 incubator (Heraeus, Germany). The selected follicles were washed three times in the culture medium and subsequently placed individually in ∼20 l droplets of the culture medium in 35-mm plastic tissueculture dishes. The micro-droplets were overlaid with autoclaved lightweight mineral oil pre-equilibrated with the medium over night at 39 ◦ C in 5% CO2 in air. The culture dishes were thus incubated for 6 days. The culture period of 6 days was found to be optimum from earlier studies in the laboratory (Hemamalini et al., 2003; Tamilmani et al., 2005). The day on which the PFs were placed in culture was taken as day 0 and subsequent days as day 1, day 2, etc. On day 0, the initial diameter of all the PFs selected for culture was recorded using a precalibrated ocular micrometer. After conducting preliminary experiments with several possible combinations and sequences of thyroxin and growth factors and hormones (data not shown), as well as based on the earlier experience in culturing sheep PFs (Hemamalini et al., 2003; Tamilmani et al., 2005), the following 15 treatments were selected to evaluate their effect on in vitro growth of sheep PFs and subsequent meiotic maturation of oocytes: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Control T4 (0-5) T4 (0-5) + EGF (0-5) T4 (0-5) + TGF-␣(0-5) T4 (0-5) + FSH (0-5) T4 (0-5) + FSH (0-5) + FCS (0-5) T4 (0-5) + LH (0-5) T4 (0-5) + FSH (0-5) + LH (0-5 T4 (0-5) + FSH (0-5) + LH (0-5) + FCS (0-5) T4 (0-1) + EGF (1-5) T4 (0-1) + TGF-␣(1-5) T4 (0-1) + FSH (1-5) T4 (0-1) + FSH (1-5) + FCS (1-5) T4 (0-1) + LH(1-5) T4 (0-1) + FSH (1-4) + LH (4-5)
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in size) was counted and the final diameter recorded at the end of the 6-day culture period. Antrum formation, as indicated by the eccentric movement of the oocyte within the follicle and extrusion of oocytes from the follicles, if any, were also recorded. 2.4. In vitro maturation of oocytes from in vitro cultured follicles At the end of day 6 of culture, the in vitro cultured follicles were carefully aspirated (if the oocytes were not extruded by then) using two 26G needles attached to 1 ml syringe barrels, to release the oocyte inside. The oocytes, collected from in vitro cultured follicles (spontaneously extruded or manually released), were washed three times in the in vitro maturation medium before being placed individually in ∼20 l droplets of IVM medium in 35mm tissue-culture dishes. The droplets were overlaid with autoclaved, pre-equilibrated lightweight mineral oil. These culture dishes were incubated at 39 ◦ C in 5% CO2 for 24 h in a CO2 incubator. This procedure regularly supports meiotic maturation of more than 80% of oocytes collected from the antral follicles in sheep (Rao et al., 2002). 2.5. Staining of in vitro matured oocytes At the end of the IVM period, the oocytes were denuded of cumulus cells by repeated pipetting through
TCM199B for 6 days Thyroxin 1 g ml−1 for 6 days Thyroxin 1 g ml−1 + EGF 50 ng ml−1 for 6 days. Thyroxin 1 g ml−1 + TGF-␣ 2.5 ng ml−1 for 6 days Thyroxin 1 g ml−1 + FSH 2 g ml−1 for 6 days Thyroxin 1 g ml−1 + FSH 2 g ml−1 + 10% FCS for 6 days Thyroxin 1 g ml−1 + LH 1 g ml−1 for 6 days Thyroxin 1 g ml−1 + FSH 2 g ml−1 + LH 1 g ml−1 for 6 days Thyroxin 1 g ml−1 + FSH 2 g ml−1 + LH 1 g ml−1 + 10% FCS for 6 days Thyroxin 1 g ml−1 for 1 day followed by EGF 50 ng ml−1 for 5 days Thyroxin 1 g ml−1 for 1 day followed by TGF-␣ 2.5 ng ml−1 for 5 days Thyroxin 1 g ml−1 for 1 day followed by FSH 2 g ml−1 for 5 days Thyroxin 1 g ml−1 for 1 day followed by FSH 2 g ml−1 + 10% FCS for 5 days Thyroxin 1 g ml−1 for 1 day followed by LH 1 g ml−1 for 5 days Thyroxin 1 g ml−1 for 1 day followed by FSH 2 g ml−1 for 4 days followed by LH 1 g ml−1 for 1 day
2.3. Morphological evaluation of pre-antral follicle development The morphology of each follicle was evaluated every 24 h using an inverted microscope (Leica, DMIRB, Germany). The number of PFs exhibiting growth (increase
a fine-bore glass pipette. Oocytes were then washed in Hoechst 33342 fluorescent stain solution (5 g ml−1 ) and incubated in a 50-l droplet of the same solution for 15 min at 38.5 ◦ C before being examined under fluorescent light on an inverted microscope for nuclear maturation to metaphase II (MII) (Rao et al., 2002).
NIL 14.00 ± 4.7a (4) NIL NIL 22.9 ± 4.3b (8) 13.3 ± 4.6a (4) NIL NIL NIL NIL NIL 20.0 ± 5.5b (6) NIL NIL NIL NIL 26.6 ± 2.4b (8) 16.6 ± 6.1 cde (6) 13.30 ± 5.05 cd (4) 37.1 ± 6.9f (13) 36.7 ± 4.3 f (11) 16.7 ± 4.3cg (5) 23.52 ± 2.0abeh (8) 24.2 ± 4.8ab (8) 19.3 ± 3.9cdeh (6) 14.2 ± 7.4c (4) 33.3 ± 4.8abde (10) 34.4 ± 4.8abh (11) 10.0 ± 4.4g (3) 21.4 ± 6.5abeh (5) 3.3a (2) 2.9c (10) 2.1ab (8) 4.36ab (5) 3.2e (14) 1.8cd (11) 3.4ab (6) 3.9c (13) 5.0c (10) 3.3ab (9) 3.4a (6) 6.9de (13) 2.7cd (13) 3.2a (5) 6.5bc (9) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Figures with same superscript(s) within a column do not differ significantly.
5.0 34.0 13.3 15.0 42.5 36.4 20.7 30.7 33.5 16.4 16.4 41.2 39.6 16.4 32.5 4.2a (2) 5.9cd (10) 5.5abf (8) 4.33abf (5) 3.3g (14) 7.8dg (11) 5.5bcf (6) 3.5cdg (13) 3.0cdg (10) 5.4bf (9) 3.6af (6) 5.1g (13) 5.1g (13) 6.2abf (5) 4.7bcf (9) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.7 33.3 26.7 16.7 40.0 36.7 20.0 38.2 30.3 26.6 21.4 43.3 40.6 16.7 32.1 Control (30) T4 (0-5) (30) T4 (0-5) + EGF (0-5) (30) T4 (0-5) + TGF-␣ (0-5) (30) T4 (0-5) + FSH (0-5) (35) T4 (0-5) + FSH (0-5) + FCS (0-5) (30) T4 (0-5) + LH (0-5) (30) T4 (0-5) + FSH (0-5) + LH (0-5) (34) T4 (0-5) + FSH (0-5) + LH (0-5) + FCS (0-5) (33) T4 (0-1) + EGF (1-5) (31) T4 (0-1) + TGF-␣ (1-5) (28) T4 (0-1) + FSH (1-5) (30) T4 (0-1) + FSH (1-5) + FCS (1-5) (32) T4 (0-1) + LH (1-5) (30) T4 (0-1) + FSH (1-4) + LH (4-5) (28)
Proportion (%) of cultured PF’s extruding oocytes Mean ± S.E. (n) Proportion of PF’s with antrum (n) Increase in diameter () of PF’s Mean ± S.E. (n)
The effects of culturing small and large PFs in different supplemented culture media are presented in Tables 1 and 2. Culture of small and large PFs in T4(0-5) + FSH(0-5) resulted in significantly better growth of PFs, a higher increase in follicular diameter, development of antrum in higher proportion of follicles, extrusion of oocytes from a larger number of cultured follicles and a higher frequency of nuclear maturation of oocytes to the MII stage (Tables 1 and 2). In the small follicles, T4(0-5) + FSH(0-5) supported a significantly higher proportion of follicles developing an antrum in culture and subsequent maturation of oocytes to metaphase II compared to T4(0-1) + FSH(1-5) (Table 1). In the large follicles, however, the difference between these two treatments was significant only with respect to extrusion of oocytes from the follicles during culture and maturation of oocytes to the MII stage (Table 2). Inclusion of LH in the culture medium for both the small and large PFs resulted in a relatively poor performance of the PFs – similar to the control medium in several respects (Tables 1 and 2). FSH, when included along with LH, was able to improve the performance of the PFs significantly (Tables 1 and 2), although none of the oocytes from small follicles cultured in media containing FSH and LH (Table 1) and oocytes from large follicles cultured in T4(0-1) + FSH(1-4) + LH(4-5) developed to the MII stage (Table 2). The performance of small PFs cultured in media containing EGF and TGF-␣ was similar to the culture in T4 (Table 1). Similar results were obtained when the data were analysed after combining the results of sequential and simultaneous treatment with the same hormones and/or
Proportion (%) of PF’s exhibiting growth Mean ± S.E. (n)
3. Results
Table 1 Influence of simultaneous and sequential treatment with hormones and growth factors on in vitro development of small PF’s in sheep
A two-factor ANOVA was performed, following arc sine transformation of the data, confirmed that the difference between types of treatments – simultaneous or sequential use of hormones and growth factors, and the treatment × type of treatment interaction – were not significant. As a follow up, the treatment effects (both simultaneous and sequential) on various parameters were subjected to one- factor ANOVA followed by Duncan’s multiple comparison test (Tables 1 and 2). Furthermore, a one-factor ANOVA, followed by Duncan’s multiple comparison test, was also conducted after combining the results of simultaneous and sequential treatment with same growth factors/hormones (Tables 3 and 4). SPSS 13.0 was used for the statistical analyses (SAS Inc., Cary, USA).
Proportion (%) of oocytes in MII Mean ± S.E.
2.6. Statistical analysis
NIL NIL NIL NIL 15.0 ± 6.3a 10.0 ± 05.3b NIL NIL NIL NIL NIL 10.0 ± 06.2b 10.0 ± 06.2b NIL NIL
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Treatments(days) (follicles)
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Table 2 Influence of simultaneous and sequential treatment with hormones and growth factors on in vitro development of large PF’s in sheep Proportion (%) of PF’s exhibiting growth Mean ± S.E. (n)
Control (28) T4 (0-5) (29) T4 (0-5) + EGF (0-5) (30) T4 (0-5) + TGF-␣ (0-5) (28) T4 (0-5) + FSH (0-5) (35) T4 (0-5) + FSH (0-5) + FCS (0-5) (30) T4 (0-5) + LH (0-5) (32) T4 (0-5) + FSH (0-5) + LH (0-5) (30) T4 (0-5) + FSH (0-5) + LH (0-5) + FCS (0-5) (28) T4 (0-1) + EGF (1-5) (29) T4 (0-1) + TGF-␣ (1-5) (28) T4 (0-1) + FSH (1-5) (32) T4 (0-1) + FSH (1-5) + FCS (1-5) (30) T4 (0-1) + LH (1-5) (30) T4 (0-1) + FSH (1-4) + LH (4-5) (28)
7.1 27.6 23.3 17.8 37.1 36.7 15.6 36.7 21.4 20.6 14.2 46.9 33.3 10.0 28.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Increase in diameter () of PF’s Mean ± S.E. (n)
04.6a (2) 05.9cdef (8) 6.3bcdef (7) 04.6abc (5) 7.0f (13) 04.3def (11) 04.3abcd (5) 03.4def (11) 04.8cdef (6) 3.6bcdef (6) 4.8abcd (4) 4.9f (15) 04.8cdf (10) 4.4ab (3) 03.8cdef (8)
7.8 33.6 13.6 12.9 41.3 35.4 13.6 26.1 30.0 15.7 8.5 48.9 34.6 10.0 23.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.3a (2) 04.7c (8) 4.9ab (7) 4.5b (5) 2.8c (13) 1.8c (11) 01.4ab (5) 05.5c (13) 4.7c (6) 3.2ab (5) 3.2ab (4) 4.8c (15) 1.4c (10) 1.1b (3) 04.7c (8)
Proportion of PF’s with antrum (n)
Proportion (%) of cultured PF’s extruding oocytes Mean ± S.E. (n)
Proportion (%) of oocytes in MII Mean ± S.E.
NIL 44.8 ± 9.3bcd (13) 16.6 ± 4.3bc (5) 10.7 ± 5.1b (3) 65.7 ± 4.0f (23) 70.0 ± 4.1ef (21) 15.6 ± 5.2bc (5) 40.0 ± 4.2bcd (12) 50.0 ± 5.5bcde (14) 13.7 ± 5.1b (4) 32.1 ± 4.6cd (9) 59.4 ± 6.9cdef (19) 46.7 ± 4.2def (14) 13.3 ± 4.8ab (4) 42.9 ± 7.1bcd (12)
NIL 24.1 ± 5.5a (7) NIL NIL 60.0 ± 5.1d (21) 53.3 ± 5.8d (16) NIL 23.3 ± 2.3a (7) 28.6 ± 6.5ab (8) NIL NIL 43.8 ± 9.3c (14) 40.0 ± 4.2c (12) NIL 28.6 ± 2.8ab (8)
NIL 15.0 ± 06.3b NIL NIL 55.0 ± 05.6d 40.0 ± 06.2c NIL 10.0 ± 05.2a 10.0 ± 04.2a NIL NIL 25.0 ± 07.7b 25.0 ± 05.3b NIL NIL
Figures with same superscript(s) within a column do not differ significantly.
Table 3 Effect of various hormones and growth factors on in vitro development of small PF’s in sheep Treatments (follicles)
Proportion (%) of PF’s exhibiting growth Mean ± S.E. (n)
Control (30) T4 (0-5) (30) T4 + EGF (61) T4 + TGF-␣ (58) T4 + FSH (65) T4 + FSH + FCS (62) T4 + LH (60) T4 + LH + FSH (62) T4 + FSH + LH + FCS (33)
6.7 33.3 26.8 18.9 41.3 38.1 15.7 35.6 30.3
± ± ± ± ± ± ± ± ±
4.2a (2) 5.9cd (10) 3.6bc (17) 2.8b (11) 3.0d (27) 4.5cd (24) 4.1ab (10) 3.1cd (22) 7.8cd (10)
Increase in diameter () of PF’s Mean ± S.E. (n) 05.0 34.0 15.0 16.4 41.1 37.5 17.7 31.4 33.5
± ± ± ± ± ± ± ± ±
0.0a (2) 2.9c (10) 1.4b (17) 1.4b (11) 2.5b (27) 2.4c (24) 2.1b (10) 2.1c (22) 3.3c (10)
Proportion of PF’s with antrum (n)
% Cultured PF’s extruding oocytes Mean ± S.E. (n)
% Oocytes in MII Mean ± S.E.
NIL 26.7 ± 2.4b (8) 19.3 ± 3.2a (12) 17.9 ± 3.6a (8) 37.3 ± 3.3c (24) 36.7 ± 3.5c (22) 15.0 ± 3.3a (9) 23.4 ± 4.2b (14) 23.0 ± 5.6b (8)
NIL 14.0 ± 4.7a (4) NIL NIL 23.7 ± 4.0b (15) 13.3 ± 4.6a (8) NIL NIL NIL
NIL NIL NIL NIL 11.0 ± 4.9a 09.0 ± 3.9a NIL NIL NIL
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Treatments (days) (follicles)
Figures with same superscript(s) within a column do not differ significantly.
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% Oocytes in MII Mean ± S.E.
NIL 15.0 ± 6.3a NIL NIL 47.9 ± 6.2c 32.6 ± 4.5b NIL 04.2 ± 4.2a 10.0 ± 4.2a NIL 23.1 ± 2.1a (7) NIL NIL 52.2 ± 4.5b (35) 46.4 ± 3.9b (28) NIL 25.5 ± 3.4a (15) 28.6 ± 6.5a (8)
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% Cultured PF’s extruding oocytes Mean ± S.E. (n)
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growth factors (Tables 3 and 4). Although a certain amount of growth could be observed in small follicles cultured in different supplemented media and the control, oocytes from small PFs cultured in T4 + FSH and T4 + FSH + FCS only matured to the MII stage (Table 3). Inclusion of TGF-␣, EGF or LH along with T4 in the culture medium for the small PFs resulted in a relatively poor performance (Table 3). Interestingly, when FSH was included along with LH, the performance of the small PFs improved, although none of the oocytes from these groups matured to the MII stage (Table 3). Performance of the large PFs was similar, although only a small proportion of oocytes from the groups cultured in FSH + LH and T4 alone matured to the MII stage.
NIL 46.4 ± 6.9bc (13) 15.3 ± 4.3a (9) 12.1 ± 3.1a (7) 62.7 ± 5.7c (42) 58.2 ± 4.3c (35) 13.8 ± 3.4a (9) 39.7 ± 03.7b (24) 50.0 ± 5.5bc (14) 05.3a (2) 0.0b (8) 1.2a (13) 2.0a (9) 2.0c (28) 1.4b (21) 1.3b (8) 2.8b (19) 4.7b (6) ± ± ± ± ± ± ± ± ± Figures with same superscript(s) within a column do not differ significantly.
7.8 33.9 14.2 11.1 46.1 35.5 11.9 26.0 30.8 4.2a (2) 2.2cd (8) 3.8bc (13) 3.3ab (9) 2.6f (28) 3.6cd (21) 2.9b (8) 2.4cd (19) 5.1 b d (6) ± ± ± ± ± ± ± ± ± 6.4 29.0 22.0 16.1 41.8 35.0 12.9 32.8 21.4 Control (28) T4 (0-5) (29) T4 + EGF (59) T4 + TGF-␣ (56s) T4 + FSH (67) T4 + FSH + FCS (60) T4 + LH (62) T4 + LH + FSH (58) T4 + FSH + LH + FCS (28)
Increase in diameter () of PF’s Mean ± S.E. (n) Proportion (%) of PF’s exhibiting growth Mean ± S.E. (n) Treatments (follicles)
Table 4 Effect of various hormones and growth factors on in vitro development of large PF’s in sheep
Proportion of PF’s with antrum (n)
4. Discussion It has been observed previously that FSH, TGF-␣ and EGF were able to induce in vitro growth of sheep PFs (Hemamalini et al., 2003) and also yield a small proportion of meiotically competent oocytes (Tamilmani et al., 2005). It is known that several endocrine, paracrine and autocrine factors are involved simultaneously and/or in a specific sequence during the complex process of ovarian folliculogenesis (Demeestere et al., 2005). Although the influence of thyroxin on ovarian folliculogenesis is well known (Dickson, 1996), its effect on in vitro development of PFs of domestic animals has never been investigated Thus it was hypothesized that treatment with thyroxine in combination or in sequence with FSH, LH, TGF-␣ and EGF may improve in vitro development of sheep PFs and the yield of meiotically competent oocytes. Thyroxin (T4 ) alone supported maturation to the MII stage in a small proportion of oocytes from cultured large PFs (Table 4). Further T4 , in combination with FSH, supported better in vitro growth, antrum development and subsequent maturation of oocytes to the MII stage in both the small and large PFs. Clearly, both thyroxin and FSH promote in vitro development of sheep PFs. However, it was previously reported (Cecconi et al., 2004) that inclusion of the thyroid hormone T3 in supraphysiological concentrations in the culture medium for mouse PFs had an adverse influence on the growth and subsequent maturation of oocytes. In addition to species differences, there are other reasons for this discrepancy. In the present study, T4, a circulatory prohormone of T3, was used, whereas Cecconi et al. (2004) used T3 . Also, the concentration of T4 (1 g/ml), used in the present study, was less than the normal physiological concentration in the circulation of sheep (Reap et al., 1978). Furthermore, the influence of the thyroxin on PFs appears to depend on the physiological status of the PFs at the
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time of collection. For example, in the present study, the response of large follicles to thyroxin was different (greater) from that of the small PFs (Tables 3 and 4). Thus, the physiological status of the mouse PFs used in the study of Cecconi et al. (2004) could also be different. It can also be seen from Tables 1–4 that the presence of LH in the culture medium invariably negated, at least in part, the positive influence of the presence of thyroxin and FSH on in vitro development of sheep PFs. This is an interesting observation. In an earlier study (Tamilmani et al., 2005), TCM199 supplemented with different levels of LH caused degeneration of sheep PFs. Further to the present study in an earlier study also (Tamilmani et al., 2005), LH was not needed, at least in a certain proportion of follicles, to induce the release of oocytes from cultured PFs in vitro. This is in contradiction to the well-known requirement of LH for ovulation in vivo. Apparently, in vitro development of sheep PFs could be achieved independent of LH. As LH was included in the in vitro maturation medium, for the oocytes collected from in vitro cultured PFs, it was not possible to indicate whether this could also be achieved independent of LH. Tamilmani et al. (2005) obtained meiotically competent oocytes for the first time from small and large PFs in sheep cultured in vitro in TGF-␣– or FSH-supplemented media. In the present study, the yield of meiotically competent oocytes from large PFs cultured in T4 + FSH was higher. Thus, the use of thyroxin clearly improved the in vitro growth and yield of meiotically competent oocytes from the cultured sheep PFs. Though, individually, TGF-␣ and FSH were able to support the in vitro development of PFs and yield meiotically competent oocytes in an earlier study (Tamilmani et al., 2005), in the present study, the use of TGF-␣ along with T4 was found to be ineffective. It is not clear why the addition of T4 annulled the positive influence of TGF-␣, although several possibilities, including the down-regulation of TGF-␣ receptors by T4 , competition for the isoreceptors between T4 and TGF-␣ and chemical antagonism between T4 and TGF-␣ need further investigation. In the present investigation, the proportion of oocytes in the GV stage at the onset of 24 h of in vitro maturation was not recorded. Therefore, it is not known what proportion of oocytes was competent to undergo in vitro maturation. Such estimates would be better indicators of the success of in vitro culture of PFs. It may be argued that failure of PFs to develop in some groups may be due to accumulation metabolic end-products as the culture medium was not replenished during the culture period of 6 days. Two lines of evidence refute this argument: (i) this 6-day culture system
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was successfully employed for sheep PFs earlier without any degeneration of the follicles (Hemamalini et al., 2003 and Tamilmani et al., 2005); and (ii) differential rates of development of PFs in different groups could not have been obtained if the PFs were degenerating due to accumulation of metabolic end-products. It can be concluded that the in vitro development of sheep PFs and the yield of meiotically competent oocytes from the cultured PFs could be significantly improved by the inclusion of thyroxin and FSH in the culture medium. Acknowledgements Department of Biotechnology (DBT), Government of India supported this work through a research grant to VHR. R. Vagdevi, B.S. Rao and B.R. Naik were supported by the DBT. G. Arunakumari received a scholarship from the Government of Andhra Pradesh, India. References Boland, N.I., Humpherson, P.G., Leese, J.H., Gosden, R.G., 1993. Pattern of lactate production and steroidogenesis during growth and maturation of mouse ovarian follicles in vitro. Biol. Reprod. 48, 798–806. Braw-Tal, R., Yossefi, S., 1997. Studies in vivo and in vitro on the initiation of follicle growth on the bovine ovary. J. Reprod. Fertil. 109, 165–171. Carroll, J., Whittingham, D.G., Wood, M.J., Telfer, E., Gosden, R.G., 1990. Extra ovarian production of mature viable mouse oocytes from frozen primary follicles. J. Reprod. Fertil. 90, 321–327. Cecconi, S., Barboni, B., Coccia, M., Mattioli, M., 1999. In vitro development of sheep preantral follicles. Biol. Reprod. 60, 594–601. Cecconi, S., Rossi, G., Coticchio, G., Macchiarelli, G., Borini, A., Canipari, R., 2004. Influence of thyroid hormone on mouse preantral follicles development in vitro. Fertil. Steril. 81 (Suppl.), 919–924. Chelikani, P.K., Amarnath, D., Reddy, K.K., Naidu, K.S., Rao, K.V., Rao, V.H., 1998. Isolation of preantral follicles in sheep and goats. Theriogenology 49, 343. Demeestere, I., Centner, J., Gervy, C., englert, Y., Delbaere, A., 2005. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction 130, 147–156. Dickson, W.M., 1996. Endocrinology. In: Swenson, M.J., Reece, W.O. (Eds.), Reproduction and Lactation. Ducke’s Physiology of Domestic Animals. Panima Publishing Corporation, New Delhi, pp. 629–710. Eppig, J.J., Schroeder, A.C., 1989. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol. Reprod. 41, 268–276. Eppig, J.J., O’Brien, M.J., 1996. Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod. 54, 197–207. Gutierrez, C.G., Ralph, J.H., Telfer, E.E., Wilmut, I., Webb, R., 2000. Growth and antrum formation of bovine preantral follicles in longterm culture in vitro. Biol. Reprod. 62, 1322–1328.
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Hemamalini, N.C., Rao, B.S., Tamilmani, G., Amarnath, D., Vagdevi, R., Naidu, K.S., Reddy, K.K., Rao, V.H., 2003. Influence of transforming growth factor-␣, insulin like growth factor-II, epidermal growth factor or follicle stimulating hormone on in vitro development of preantral follicles in sheep. Small Rumin. Res. 50, 11–22. Hirao, Y., Nagai, T., Kubo, M., Miyano, T., Miyake, M., Kato, S., 1994. In vitro growth and maturation of pig oocytes. J. Reprod. Fertil. 101, 333–339. Liu, J., Van der Elst, J., Van den Broecke, R., Dhont, M., 2001. Live offspring by in vitro fertilization of oocytes from cryopreserved primoridal mouse follicles after sequential in vivo transplantation and in vitro maturation. Biol. Reprod. 64, 171–178. Mao, J., Wu, G., Smith, M.F., McCauley, T.C., Cantley, T.C., Prather, R.S., Didion, B.A., Day, B.N., 2002. Effects of culture medium, serum type, and various concentrations of follicle stimulating hormone on procine preantral follicle development and antrum formation in vitro. Biol. Reprod. 67, 1197–1203. Nayudu, P.L., Osborn, S.M., 1992. Factors influencing the rate of preantral and antral growth of mouse ovarian follicles in vitro. J. Reprod. Fertil. 95, 349–362.
Nuttinck, F., Mermillod, P., Massip, A., Dessy, F., 1993. Characterization of in vitro growth of bovine preantral ovarian follicles: a preliminary study. Theriogenology 39, 811–821. Rajarajan, K., Rao, B.S., Vagdevi, R., Tamilmani, G., Arunakumari, G., Sreenu, M., Amarnath, D., Naik, B.R., Rao, V.H., 2006. Influence of various growth factors on in vitro development of goat preantral follicles. Small Rumin. Res. 63, 204–212. Rao, B.S., Naidu, K.S., Amarnath, D., Vagdevi, R., Rao, A.S., Bramaiah, K.V., Rao, V.H., 2002. In vitro maturation of sheep oocytes in different media during breeding and non-breeding seasons. Small Rumin. Res. 43, 31–36. Reap, M., Cass, C., Hightower, D., 1978. Thyroxine and tiriiodo thyronine levels in ten species of animals. Southwestern Vet. 31, 31. Sviridov, B.E., Kaganskaya, V.V., 1991. A quantitative study of the development of preantral follicles of cattle during in vitro culture. Vestnik-Sel’skhokhozyaistvennoi-Nauki-Moskva 6, 112–117. Tamilmani, G., Rao, B.S., Vagdevi, R., Amarnath, D., Naik, B.R., Mutharao, M., Rao, V.H., 2005. Nuclear maturation of oocytes in sheep preantral follilces cultured in vitro. Small Rumin. Res. 60, 295–305.
Effect of hormones and growth factors on in vitro development of sheep preantral follicles G. Arunakumari a , R. Vagdevi a , B.S. Rao a,1 , B.R. Naik a , K.S. Naidu a,2 , R.V. Suresh Kumar a,b , V.H. Rao a,∗ a
Embryo Biotechnology Laboratory, Department of Physiology, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati 517502, India b Department of Surgery and Radiology, India
Received 19 January 2005; received in revised form 4 January 2006; accepted 4 January 2006 Available online 3 April 2006
Abstract The present investigation attempts to improve the frequency of in vitro maturation of oocytes by culturing small (150–250 m) and large (>250–400 m) preantral follicles (PFs) of sheep for 6 days in various combinations/sequences of thyroxin (T4 ), FSH, LH, transforming growth factor alpha (TGF-␣), epidermal growth factor (EGF) and heat-treated foetal calf serum (FCS). Bicarbonatebuffered tissue culture medium 199, supplemented with 50 g ml−1 gentamicin sulphate, served as the control medium. In vitro development was initially assessed by the proportion of PFs exhibiting an increase in size, mean increase in diameter and antrum formation. Nuclear maturation to the metaphase II stage of the oocytes isolated from cultured PFs, after an additional 24-h in vitro maturation, indicated success. A total of 15% of oocytes from small PFs and 55% from large PFs, cultured in T4 + FSH, matured to metaphase II. Culture of PFs in other combinations/sequences of hormones and growth factors, including the control medium, supported a significantly lower proportion of oocytes maturing to metaphase II stage. It is concluded that 6-day in vitro culture of sheep PFs in thyroxin and FSH greatly improves the frequency of oocyte maturation to metaphase II stage. © 2006 Elsevier B.V. All rights reserved. Keywords: Sheep; Preantral follicles; Thyroxin; Growth factors; FSH
1. Introduction Biotechnology tools, such as embryo transfer, embryo cryopreservation and in vitro production of embryos, have only enhanced the utilisation of female genetic
∗ Corresponding author. Tel.: +91 877 2249376; fax: +91 877 2249563. E-mail address: [email protected] (V.H. Rao). 1 Present address: Center for Cellular and Molecular Biology, Hyderabad, India. 2 Present address: Department of Animal Reproduction and Gynecology, India.
0921-4488/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2006.01.002
resources to a limited extent, as full advantage is not taken of the large number of potential oocytes present in primordial and preantral follicles (PFs) in the ovaries. Most of the oocytes in PFs never become available for ovulation and fertilization. Therefore, to maximize the utilization of female gametes, repeatable in vitro techniques for maturing PFs and subsequent fertilization of oocytes are desirable. Other applications of in vitro maturation of PFs have been detailed previously (Mao et al., 2002; Hemamalini et al., 2003; Demeestere et al., 2005; Tamilmani et al., 2005). Only in the mouse has in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes from PFs resulted
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in the birth of live offspring (Nayudu and Osborn, 1992; Boland et al., 1993; Eppig and Schroeder, 1989; Carroll et al., 1990; Eppig and O’Brien, 1996; Liu et al., 2001). Follicular antrum development in cultured PFs of pig (Hirao et al., 1994) and cow (Sviridov and Kaganskaya, 1991; Nuttinck et al., 1993; Braw-Tal and Yossefi, 1997; Gutierrez et al., 2000) has also been accomplished. In the pig, embryos were produced from oocytes collected from cultured PFs (Wu et al., 2001). Chelikani et al. (1998) reported the isolation of PFs from sheep and goat ovaries for the first time. Antrum development in cultured sheep PFs has also been reported (Cecconi et al., 1999), while success in the growth and new DNA synthesis in cultured sheep and goat PFs was only recently achieved (Hemamalini et al., 2003; Rajarajan et al., 2006). Recently, 30% of oocytes from sheep PFs cultured in TGF-␣ or FSH could be matured to MII stage for the first time (Tamilmani et al., 2005). However, meiotic maturation rates in excess of 80% for oocytes isolated from the antral follicles of sheep have been reported earlier (Rao et al., 2002). The reported success in meiotic maturation of sheep oocytes from cultured PFs needs to be improved, however (Tamilmani et al., 2005). Several endocrine, paracrine and autocrine factors are involved simultaneously and/or in a specific sequence during the complex process of ovarian folliculogenesis (Demeestere et al., 2005). Although the importance of thyroxin in the growth and development of ovarian follicles is well known (Dickson, 1996), it has never been used in the culture medium for PFs in domestic animals. Thus, the goal of the present study was to improve the meiotic maturation rate of oocytes by in vitro culture of sheep PFs in different combinations/sequences of thyroxin, FSH, LH, EGF and TGF-alpha. 2. Materials and methods Unless otherwise stated, all culture media, hormones, growth factors, fetal calf serum (FCS) and chemicals were purchased from the Sigma (St. Louis, MO, USA) and plastics from Nunclon (Roskilde, Denmark). Barring the media supplemented with different growth factors and hormones all other solutions were filtered through a 0.22-m sterilizing filter (Sartorius, Germany) prior to use. All media were incubated at 39 ◦ C under a humidified atmosphere of 5% CO2 in air for 1 h prior to use.
tissue culture medium 199 (TCM199H), supplemented with 25 IU ml−1 heparin and 50 g ml−1 gentamicin sulphate], 0.5% bovine serum albumin (BSA), stock solutions of TGF-␣, EGF, FSH and LH have all been described previously (Tamilmani et al., 2005). The source, potency and endotoxin levels for the different growth factors and hormones used in this study were the same as those described by Tamilmani et al. (2005). All the stock solutions were stored at −20 ◦ C until used. Thyroxin stock solution was prepared by dissolving a 100-g thyroxin sodium tablet (IP) (ELTROXIN, Glaxo India Limited) in 10 ml bicarbonate-buffered tissue culture medium 199 (TCM199B), supplemented with 50 g ml−1 gentamicin sulphate, to yield a concentration of 10 g ml−1 . Thyroxin-supplemented culture medium was prepared by diluting 500 l of the thyroxin stock solution in 4500 l of control medium to obtain a final concentration of 1 g ml−1 thyroxin (T4 ). Supplementation of the culture medium with one or more of the other hormones and growth factors was achieved by appropriately mixing thyroxinsupplemented medium with appropriate volumes of the stock solutions. For example, supplementation of the culture medium with thyroxin 1 g ml−1 and TGF-␣ 2.5 ng ml−1 was achieved by adding 1995 l thyroxinsupplemented culture medium to 5 l of the TGF-␣ stock solution. Similarly, supplementation of the culture medium with thyroxin 1 g ml−1 , FSH 2 g ml−1 and LH 1 g ml−1 was achieved by mixing 1970 l of thyroxin-supplemented medium, 28 l FSH stock solution and 2 l LH stock solution. Various supplemented media were stored for up to 1 week at 4 ◦ C in a disposable syringe and used directly without filtering. TCM199B, supplemented with 50 g ml−1 gentamicin sulphate, was used as the control medium. This was filtered through a 0.22-m filter and stored at 4 ◦ C for up to 1 week. TCM199B, supplemented with 0.5 g ml−1 FSH, 100 g ml−1 LH, 1 g ml−1 estradiol 17, 50 g ml−1 gentamicin sulphate, 10 g ml−1 BSA and 20% (v/v) heat-treated oestrus sheep serum was used for the in vitro maturation (IVM) of oocytes collected from the cultured PFs (Rao et al., 2002). This was stored at 4 ◦ C for up to 1 week and equilibrated for 1 h at 39 ◦ C in 5% CO2 in air, prior to use. 2.2. Collection of ovaries, isolation, selection and culture of PFs
2.1. Culture media Preparation of phosphate-buffered saline (PBS), the handling medium for PFs and oocytes [HEPES-buffered
Collection of ovaries, isolation of PFs, classification into small and large sizes, selection of PFs for cultures were performed, as described by Tamilmani et al. (2005).
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All the small and large follicles isolated on any day were randomly allotted to one of the 15 treatments and each treatment was replicated seven times. Culture procedures for PFs were followed, as standardized previously (Hemamalini et al., 2003; Tamilmani et al., 2005). Briefly, the appropriate culture medium was pre-incubated for 1 h at 39 ◦ C in 5% CO2 in air in a CO2 incubator (Heraeus, Germany). The selected follicles were washed three times in the culture medium and subsequently placed individually in ∼20 l droplets of the culture medium in 35-mm plastic tissueculture dishes. The micro-droplets were overlaid with autoclaved lightweight mineral oil pre-equilibrated with the medium over night at 39 ◦ C in 5% CO2 in air. The culture dishes were thus incubated for 6 days. The culture period of 6 days was found to be optimum from earlier studies in the laboratory (Hemamalini et al., 2003; Tamilmani et al., 2005). The day on which the PFs were placed in culture was taken as day 0 and subsequent days as day 1, day 2, etc. On day 0, the initial diameter of all the PFs selected for culture was recorded using a precalibrated ocular micrometer. After conducting preliminary experiments with several possible combinations and sequences of thyroxin and growth factors and hormones (data not shown), as well as based on the earlier experience in culturing sheep PFs (Hemamalini et al., 2003; Tamilmani et al., 2005), the following 15 treatments were selected to evaluate their effect on in vitro growth of sheep PFs and subsequent meiotic maturation of oocytes: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Control T4 (0-5) T4 (0-5) + EGF (0-5) T4 (0-5) + TGF-␣(0-5) T4 (0-5) + FSH (0-5) T4 (0-5) + FSH (0-5) + FCS (0-5) T4 (0-5) + LH (0-5) T4 (0-5) + FSH (0-5) + LH (0-5 T4 (0-5) + FSH (0-5) + LH (0-5) + FCS (0-5) T4 (0-1) + EGF (1-5) T4 (0-1) + TGF-␣(1-5) T4 (0-1) + FSH (1-5) T4 (0-1) + FSH (1-5) + FCS (1-5) T4 (0-1) + LH(1-5) T4 (0-1) + FSH (1-4) + LH (4-5)
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in size) was counted and the final diameter recorded at the end of the 6-day culture period. Antrum formation, as indicated by the eccentric movement of the oocyte within the follicle and extrusion of oocytes from the follicles, if any, were also recorded. 2.4. In vitro maturation of oocytes from in vitro cultured follicles At the end of day 6 of culture, the in vitro cultured follicles were carefully aspirated (if the oocytes were not extruded by then) using two 26G needles attached to 1 ml syringe barrels, to release the oocyte inside. The oocytes, collected from in vitro cultured follicles (spontaneously extruded or manually released), were washed three times in the in vitro maturation medium before being placed individually in ∼20 l droplets of IVM medium in 35mm tissue-culture dishes. The droplets were overlaid with autoclaved, pre-equilibrated lightweight mineral oil. These culture dishes were incubated at 39 ◦ C in 5% CO2 for 24 h in a CO2 incubator. This procedure regularly supports meiotic maturation of more than 80% of oocytes collected from the antral follicles in sheep (Rao et al., 2002). 2.5. Staining of in vitro matured oocytes At the end of the IVM period, the oocytes were denuded of cumulus cells by repeated pipetting through
TCM199B for 6 days Thyroxin 1 g ml−1 for 6 days Thyroxin 1 g ml−1 + EGF 50 ng ml−1 for 6 days. Thyroxin 1 g ml−1 + TGF-␣ 2.5 ng ml−1 for 6 days Thyroxin 1 g ml−1 + FSH 2 g ml−1 for 6 days Thyroxin 1 g ml−1 + FSH 2 g ml−1 + 10% FCS for 6 days Thyroxin 1 g ml−1 + LH 1 g ml−1 for 6 days Thyroxin 1 g ml−1 + FSH 2 g ml−1 + LH 1 g ml−1 for 6 days Thyroxin 1 g ml−1 + FSH 2 g ml−1 + LH 1 g ml−1 + 10% FCS for 6 days Thyroxin 1 g ml−1 for 1 day followed by EGF 50 ng ml−1 for 5 days Thyroxin 1 g ml−1 for 1 day followed by TGF-␣ 2.5 ng ml−1 for 5 days Thyroxin 1 g ml−1 for 1 day followed by FSH 2 g ml−1 for 5 days Thyroxin 1 g ml−1 for 1 day followed by FSH 2 g ml−1 + 10% FCS for 5 days Thyroxin 1 g ml−1 for 1 day followed by LH 1 g ml−1 for 5 days Thyroxin 1 g ml−1 for 1 day followed by FSH 2 g ml−1 for 4 days followed by LH 1 g ml−1 for 1 day
2.3. Morphological evaluation of pre-antral follicle development The morphology of each follicle was evaluated every 24 h using an inverted microscope (Leica, DMIRB, Germany). The number of PFs exhibiting growth (increase
a fine-bore glass pipette. Oocytes were then washed in Hoechst 33342 fluorescent stain solution (5 g ml−1 ) and incubated in a 50-l droplet of the same solution for 15 min at 38.5 ◦ C before being examined under fluorescent light on an inverted microscope for nuclear maturation to metaphase II (MII) (Rao et al., 2002).
NIL 14.00 ± 4.7a (4) NIL NIL 22.9 ± 4.3b (8) 13.3 ± 4.6a (4) NIL NIL NIL NIL NIL 20.0 ± 5.5b (6) NIL NIL NIL NIL 26.6 ± 2.4b (8) 16.6 ± 6.1 cde (6) 13.30 ± 5.05 cd (4) 37.1 ± 6.9f (13) 36.7 ± 4.3 f (11) 16.7 ± 4.3cg (5) 23.52 ± 2.0abeh (8) 24.2 ± 4.8ab (8) 19.3 ± 3.9cdeh (6) 14.2 ± 7.4c (4) 33.3 ± 4.8abde (10) 34.4 ± 4.8abh (11) 10.0 ± 4.4g (3) 21.4 ± 6.5abeh (5) 3.3a (2) 2.9c (10) 2.1ab (8) 4.36ab (5) 3.2e (14) 1.8cd (11) 3.4ab (6) 3.9c (13) 5.0c (10) 3.3ab (9) 3.4a (6) 6.9de (13) 2.7cd (13) 3.2a (5) 6.5bc (9) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Figures with same superscript(s) within a column do not differ significantly.
5.0 34.0 13.3 15.0 42.5 36.4 20.7 30.7 33.5 16.4 16.4 41.2 39.6 16.4 32.5 4.2a (2) 5.9cd (10) 5.5abf (8) 4.33abf (5) 3.3g (14) 7.8dg (11) 5.5bcf (6) 3.5cdg (13) 3.0cdg (10) 5.4bf (9) 3.6af (6) 5.1g (13) 5.1g (13) 6.2abf (5) 4.7bcf (9) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.7 33.3 26.7 16.7 40.0 36.7 20.0 38.2 30.3 26.6 21.4 43.3 40.6 16.7 32.1 Control (30) T4 (0-5) (30) T4 (0-5) + EGF (0-5) (30) T4 (0-5) + TGF-␣ (0-5) (30) T4 (0-5) + FSH (0-5) (35) T4 (0-5) + FSH (0-5) + FCS (0-5) (30) T4 (0-5) + LH (0-5) (30) T4 (0-5) + FSH (0-5) + LH (0-5) (34) T4 (0-5) + FSH (0-5) + LH (0-5) + FCS (0-5) (33) T4 (0-1) + EGF (1-5) (31) T4 (0-1) + TGF-␣ (1-5) (28) T4 (0-1) + FSH (1-5) (30) T4 (0-1) + FSH (1-5) + FCS (1-5) (32) T4 (0-1) + LH (1-5) (30) T4 (0-1) + FSH (1-4) + LH (4-5) (28)
Proportion (%) of cultured PF’s extruding oocytes Mean ± S.E. (n) Proportion of PF’s with antrum (n) Increase in diameter () of PF’s Mean ± S.E. (n)
The effects of culturing small and large PFs in different supplemented culture media are presented in Tables 1 and 2. Culture of small and large PFs in T4(0-5) + FSH(0-5) resulted in significantly better growth of PFs, a higher increase in follicular diameter, development of antrum in higher proportion of follicles, extrusion of oocytes from a larger number of cultured follicles and a higher frequency of nuclear maturation of oocytes to the MII stage (Tables 1 and 2). In the small follicles, T4(0-5) + FSH(0-5) supported a significantly higher proportion of follicles developing an antrum in culture and subsequent maturation of oocytes to metaphase II compared to T4(0-1) + FSH(1-5) (Table 1). In the large follicles, however, the difference between these two treatments was significant only with respect to extrusion of oocytes from the follicles during culture and maturation of oocytes to the MII stage (Table 2). Inclusion of LH in the culture medium for both the small and large PFs resulted in a relatively poor performance of the PFs – similar to the control medium in several respects (Tables 1 and 2). FSH, when included along with LH, was able to improve the performance of the PFs significantly (Tables 1 and 2), although none of the oocytes from small follicles cultured in media containing FSH and LH (Table 1) and oocytes from large follicles cultured in T4(0-1) + FSH(1-4) + LH(4-5) developed to the MII stage (Table 2). The performance of small PFs cultured in media containing EGF and TGF-␣ was similar to the culture in T4 (Table 1). Similar results were obtained when the data were analysed after combining the results of sequential and simultaneous treatment with the same hormones and/or
Proportion (%) of PF’s exhibiting growth Mean ± S.E. (n)
3. Results
Table 1 Influence of simultaneous and sequential treatment with hormones and growth factors on in vitro development of small PF’s in sheep
A two-factor ANOVA was performed, following arc sine transformation of the data, confirmed that the difference between types of treatments – simultaneous or sequential use of hormones and growth factors, and the treatment × type of treatment interaction – were not significant. As a follow up, the treatment effects (both simultaneous and sequential) on various parameters were subjected to one- factor ANOVA followed by Duncan’s multiple comparison test (Tables 1 and 2). Furthermore, a one-factor ANOVA, followed by Duncan’s multiple comparison test, was also conducted after combining the results of simultaneous and sequential treatment with same growth factors/hormones (Tables 3 and 4). SPSS 13.0 was used for the statistical analyses (SAS Inc., Cary, USA).
Proportion (%) of oocytes in MII Mean ± S.E.
2.6. Statistical analysis
NIL NIL NIL NIL 15.0 ± 6.3a 10.0 ± 05.3b NIL NIL NIL NIL NIL 10.0 ± 06.2b 10.0 ± 06.2b NIL NIL
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Treatments(days) (follicles)
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Table 2 Influence of simultaneous and sequential treatment with hormones and growth factors on in vitro development of large PF’s in sheep Proportion (%) of PF’s exhibiting growth Mean ± S.E. (n)
Control (28) T4 (0-5) (29) T4 (0-5) + EGF (0-5) (30) T4 (0-5) + TGF-␣ (0-5) (28) T4 (0-5) + FSH (0-5) (35) T4 (0-5) + FSH (0-5) + FCS (0-5) (30) T4 (0-5) + LH (0-5) (32) T4 (0-5) + FSH (0-5) + LH (0-5) (30) T4 (0-5) + FSH (0-5) + LH (0-5) + FCS (0-5) (28) T4 (0-1) + EGF (1-5) (29) T4 (0-1) + TGF-␣ (1-5) (28) T4 (0-1) + FSH (1-5) (32) T4 (0-1) + FSH (1-5) + FCS (1-5) (30) T4 (0-1) + LH (1-5) (30) T4 (0-1) + FSH (1-4) + LH (4-5) (28)
7.1 27.6 23.3 17.8 37.1 36.7 15.6 36.7 21.4 20.6 14.2 46.9 33.3 10.0 28.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Increase in diameter () of PF’s Mean ± S.E. (n)
04.6a (2) 05.9cdef (8) 6.3bcdef (7) 04.6abc (5) 7.0f (13) 04.3def (11) 04.3abcd (5) 03.4def (11) 04.8cdef (6) 3.6bcdef (6) 4.8abcd (4) 4.9f (15) 04.8cdf (10) 4.4ab (3) 03.8cdef (8)
7.8 33.6 13.6 12.9 41.3 35.4 13.6 26.1 30.0 15.7 8.5 48.9 34.6 10.0 23.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.3a (2) 04.7c (8) 4.9ab (7) 4.5b (5) 2.8c (13) 1.8c (11) 01.4ab (5) 05.5c (13) 4.7c (6) 3.2ab (5) 3.2ab (4) 4.8c (15) 1.4c (10) 1.1b (3) 04.7c (8)
Proportion of PF’s with antrum (n)
Proportion (%) of cultured PF’s extruding oocytes Mean ± S.E. (n)
Proportion (%) of oocytes in MII Mean ± S.E.
NIL 44.8 ± 9.3bcd (13) 16.6 ± 4.3bc (5) 10.7 ± 5.1b (3) 65.7 ± 4.0f (23) 70.0 ± 4.1ef (21) 15.6 ± 5.2bc (5) 40.0 ± 4.2bcd (12) 50.0 ± 5.5bcde (14) 13.7 ± 5.1b (4) 32.1 ± 4.6cd (9) 59.4 ± 6.9cdef (19) 46.7 ± 4.2def (14) 13.3 ± 4.8ab (4) 42.9 ± 7.1bcd (12)
NIL 24.1 ± 5.5a (7) NIL NIL 60.0 ± 5.1d (21) 53.3 ± 5.8d (16) NIL 23.3 ± 2.3a (7) 28.6 ± 6.5ab (8) NIL NIL 43.8 ± 9.3c (14) 40.0 ± 4.2c (12) NIL 28.6 ± 2.8ab (8)
NIL 15.0 ± 06.3b NIL NIL 55.0 ± 05.6d 40.0 ± 06.2c NIL 10.0 ± 05.2a 10.0 ± 04.2a NIL NIL 25.0 ± 07.7b 25.0 ± 05.3b NIL NIL
Figures with same superscript(s) within a column do not differ significantly.
Table 3 Effect of various hormones and growth factors on in vitro development of small PF’s in sheep Treatments (follicles)
Proportion (%) of PF’s exhibiting growth Mean ± S.E. (n)
Control (30) T4 (0-5) (30) T4 + EGF (61) T4 + TGF-␣ (58) T4 + FSH (65) T4 + FSH + FCS (62) T4 + LH (60) T4 + LH + FSH (62) T4 + FSH + LH + FCS (33)
6.7 33.3 26.8 18.9 41.3 38.1 15.7 35.6 30.3
± ± ± ± ± ± ± ± ±
4.2a (2) 5.9cd (10) 3.6bc (17) 2.8b (11) 3.0d (27) 4.5cd (24) 4.1ab (10) 3.1cd (22) 7.8cd (10)
Increase in diameter () of PF’s Mean ± S.E. (n) 05.0 34.0 15.0 16.4 41.1 37.5 17.7 31.4 33.5
± ± ± ± ± ± ± ± ±
0.0a (2) 2.9c (10) 1.4b (17) 1.4b (11) 2.5b (27) 2.4c (24) 2.1b (10) 2.1c (22) 3.3c (10)
Proportion of PF’s with antrum (n)
% Cultured PF’s extruding oocytes Mean ± S.E. (n)
% Oocytes in MII Mean ± S.E.
NIL 26.7 ± 2.4b (8) 19.3 ± 3.2a (12) 17.9 ± 3.6a (8) 37.3 ± 3.3c (24) 36.7 ± 3.5c (22) 15.0 ± 3.3a (9) 23.4 ± 4.2b (14) 23.0 ± 5.6b (8)
NIL 14.0 ± 4.7a (4) NIL NIL 23.7 ± 4.0b (15) 13.3 ± 4.6a (8) NIL NIL NIL
NIL NIL NIL NIL 11.0 ± 4.9a 09.0 ± 3.9a NIL NIL NIL
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Treatments (days) (follicles)
Figures with same superscript(s) within a column do not differ significantly.
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% Oocytes in MII Mean ± S.E.
NIL 15.0 ± 6.3a NIL NIL 47.9 ± 6.2c 32.6 ± 4.5b NIL 04.2 ± 4.2a 10.0 ± 4.2a NIL 23.1 ± 2.1a (7) NIL NIL 52.2 ± 4.5b (35) 46.4 ± 3.9b (28) NIL 25.5 ± 3.4a (15) 28.6 ± 6.5a (8)
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% Cultured PF’s extruding oocytes Mean ± S.E. (n)
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growth factors (Tables 3 and 4). Although a certain amount of growth could be observed in small follicles cultured in different supplemented media and the control, oocytes from small PFs cultured in T4 + FSH and T4 + FSH + FCS only matured to the MII stage (Table 3). Inclusion of TGF-␣, EGF or LH along with T4 in the culture medium for the small PFs resulted in a relatively poor performance (Table 3). Interestingly, when FSH was included along with LH, the performance of the small PFs improved, although none of the oocytes from these groups matured to the MII stage (Table 3). Performance of the large PFs was similar, although only a small proportion of oocytes from the groups cultured in FSH + LH and T4 alone matured to the MII stage.
NIL 46.4 ± 6.9bc (13) 15.3 ± 4.3a (9) 12.1 ± 3.1a (7) 62.7 ± 5.7c (42) 58.2 ± 4.3c (35) 13.8 ± 3.4a (9) 39.7 ± 03.7b (24) 50.0 ± 5.5bc (14) 05.3a (2) 0.0b (8) 1.2a (13) 2.0a (9) 2.0c (28) 1.4b (21) 1.3b (8) 2.8b (19) 4.7b (6) ± ± ± ± ± ± ± ± ± Figures with same superscript(s) within a column do not differ significantly.
7.8 33.9 14.2 11.1 46.1 35.5 11.9 26.0 30.8 4.2a (2) 2.2cd (8) 3.8bc (13) 3.3ab (9) 2.6f (28) 3.6cd (21) 2.9b (8) 2.4cd (19) 5.1 b d (6) ± ± ± ± ± ± ± ± ± 6.4 29.0 22.0 16.1 41.8 35.0 12.9 32.8 21.4 Control (28) T4 (0-5) (29) T4 + EGF (59) T4 + TGF-␣ (56s) T4 + FSH (67) T4 + FSH + FCS (60) T4 + LH (62) T4 + LH + FSH (58) T4 + FSH + LH + FCS (28)
Increase in diameter () of PF’s Mean ± S.E. (n) Proportion (%) of PF’s exhibiting growth Mean ± S.E. (n) Treatments (follicles)
Table 4 Effect of various hormones and growth factors on in vitro development of large PF’s in sheep
Proportion of PF’s with antrum (n)
4. Discussion It has been observed previously that FSH, TGF-␣ and EGF were able to induce in vitro growth of sheep PFs (Hemamalini et al., 2003) and also yield a small proportion of meiotically competent oocytes (Tamilmani et al., 2005). It is known that several endocrine, paracrine and autocrine factors are involved simultaneously and/or in a specific sequence during the complex process of ovarian folliculogenesis (Demeestere et al., 2005). Although the influence of thyroxin on ovarian folliculogenesis is well known (Dickson, 1996), its effect on in vitro development of PFs of domestic animals has never been investigated Thus it was hypothesized that treatment with thyroxine in combination or in sequence with FSH, LH, TGF-␣ and EGF may improve in vitro development of sheep PFs and the yield of meiotically competent oocytes. Thyroxin (T4 ) alone supported maturation to the MII stage in a small proportion of oocytes from cultured large PFs (Table 4). Further T4 , in combination with FSH, supported better in vitro growth, antrum development and subsequent maturation of oocytes to the MII stage in both the small and large PFs. Clearly, both thyroxin and FSH promote in vitro development of sheep PFs. However, it was previously reported (Cecconi et al., 2004) that inclusion of the thyroid hormone T3 in supraphysiological concentrations in the culture medium for mouse PFs had an adverse influence on the growth and subsequent maturation of oocytes. In addition to species differences, there are other reasons for this discrepancy. In the present study, T4, a circulatory prohormone of T3, was used, whereas Cecconi et al. (2004) used T3 . Also, the concentration of T4 (1 g/ml), used in the present study, was less than the normal physiological concentration in the circulation of sheep (Reap et al., 1978). Furthermore, the influence of the thyroxin on PFs appears to depend on the physiological status of the PFs at the
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time of collection. For example, in the present study, the response of large follicles to thyroxin was different (greater) from that of the small PFs (Tables 3 and 4). Thus, the physiological status of the mouse PFs used in the study of Cecconi et al. (2004) could also be different. It can also be seen from Tables 1–4 that the presence of LH in the culture medium invariably negated, at least in part, the positive influence of the presence of thyroxin and FSH on in vitro development of sheep PFs. This is an interesting observation. In an earlier study (Tamilmani et al., 2005), TCM199 supplemented with different levels of LH caused degeneration of sheep PFs. Further to the present study in an earlier study also (Tamilmani et al., 2005), LH was not needed, at least in a certain proportion of follicles, to induce the release of oocytes from cultured PFs in vitro. This is in contradiction to the well-known requirement of LH for ovulation in vivo. Apparently, in vitro development of sheep PFs could be achieved independent of LH. As LH was included in the in vitro maturation medium, for the oocytes collected from in vitro cultured PFs, it was not possible to indicate whether this could also be achieved independent of LH. Tamilmani et al. (2005) obtained meiotically competent oocytes for the first time from small and large PFs in sheep cultured in vitro in TGF-␣– or FSH-supplemented media. In the present study, the yield of meiotically competent oocytes from large PFs cultured in T4 + FSH was higher. Thus, the use of thyroxin clearly improved the in vitro growth and yield of meiotically competent oocytes from the cultured sheep PFs. Though, individually, TGF-␣ and FSH were able to support the in vitro development of PFs and yield meiotically competent oocytes in an earlier study (Tamilmani et al., 2005), in the present study, the use of TGF-␣ along with T4 was found to be ineffective. It is not clear why the addition of T4 annulled the positive influence of TGF-␣, although several possibilities, including the down-regulation of TGF-␣ receptors by T4 , competition for the isoreceptors between T4 and TGF-␣ and chemical antagonism between T4 and TGF-␣ need further investigation. In the present investigation, the proportion of oocytes in the GV stage at the onset of 24 h of in vitro maturation was not recorded. Therefore, it is not known what proportion of oocytes was competent to undergo in vitro maturation. Such estimates would be better indicators of the success of in vitro culture of PFs. It may be argued that failure of PFs to develop in some groups may be due to accumulation metabolic end-products as the culture medium was not replenished during the culture period of 6 days. Two lines of evidence refute this argument: (i) this 6-day culture system
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was successfully employed for sheep PFs earlier without any degeneration of the follicles (Hemamalini et al., 2003 and Tamilmani et al., 2005); and (ii) differential rates of development of PFs in different groups could not have been obtained if the PFs were degenerating due to accumulation of metabolic end-products. It can be concluded that the in vitro development of sheep PFs and the yield of meiotically competent oocytes from the cultured PFs could be significantly improved by the inclusion of thyroxin and FSH in the culture medium. Acknowledgements Department of Biotechnology (DBT), Government of India supported this work through a research grant to VHR. R. Vagdevi, B.S. Rao and B.R. Naik were supported by the DBT. G. Arunakumari received a scholarship from the Government of Andhra Pradesh, India. References Boland, N.I., Humpherson, P.G., Leese, J.H., Gosden, R.G., 1993. Pattern of lactate production and steroidogenesis during growth and maturation of mouse ovarian follicles in vitro. Biol. Reprod. 48, 798–806. Braw-Tal, R., Yossefi, S., 1997. Studies in vivo and in vitro on the initiation of follicle growth on the bovine ovary. J. Reprod. Fertil. 109, 165–171. Carroll, J., Whittingham, D.G., Wood, M.J., Telfer, E., Gosden, R.G., 1990. Extra ovarian production of mature viable mouse oocytes from frozen primary follicles. J. Reprod. Fertil. 90, 321–327. Cecconi, S., Barboni, B., Coccia, M., Mattioli, M., 1999. In vitro development of sheep preantral follicles. Biol. Reprod. 60, 594–601. Cecconi, S., Rossi, G., Coticchio, G., Macchiarelli, G., Borini, A., Canipari, R., 2004. Influence of thyroid hormone on mouse preantral follicles development in vitro. Fertil. Steril. 81 (Suppl.), 919–924. Chelikani, P.K., Amarnath, D., Reddy, K.K., Naidu, K.S., Rao, K.V., Rao, V.H., 1998. Isolation of preantral follicles in sheep and goats. Theriogenology 49, 343. Demeestere, I., Centner, J., Gervy, C., englert, Y., Delbaere, A., 2005. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction 130, 147–156. Dickson, W.M., 1996. Endocrinology. In: Swenson, M.J., Reece, W.O. (Eds.), Reproduction and Lactation. Ducke’s Physiology of Domestic Animals. Panima Publishing Corporation, New Delhi, pp. 629–710. Eppig, J.J., Schroeder, A.C., 1989. Capacity of mouse oocytes from preantral follicles to undergo embryogenesis and development to live young after growth, maturation, and fertilization in vitro. Biol. Reprod. 41, 268–276. Eppig, J.J., O’Brien, M.J., 1996. Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod. 54, 197–207. Gutierrez, C.G., Ralph, J.H., Telfer, E.E., Wilmut, I., Webb, R., 2000. Growth and antrum formation of bovine preantral follicles in longterm culture in vitro. Biol. Reprod. 62, 1322–1328.
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