Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability

Animal Reproduction Science 81 (2004) 77–86

Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability C. Accardo a,∗ , M. Dattena a , S. Pilichi a , L. Mara a , B. Chessa b , P. Cappai a a b

Istituto Zootecnico e Caseario per la Sardegna, Servizio Riproduzione Animale, 07040 Olmedo, Italy Facoltà di Medicina Veterinaria, Università degli Studi di Sassari, Via Vienna 2, 07100 Sassari, Italy Received 31 March 2003; received in revised form 11 September 2003; accepted 3 October 2003

Abstract The objective of this work was to study the effect of a preparation of human recombinant gonadotrophins (r-FSH and r-LH) on the in vitro maturation (IVM) and development of sheep oocytes. In addition, the viability of fresh and vitrified blastocysts obtained after transfer was tested. Oocytes collected from slaughtered animals were divided into five different maturation groups. All groups were matured in a medium containing TCM199 with 4 mg/ml BSA, 100 ␮M cysteamine and 1 ␮g/ml estradiol-17␤. Each group was also treated with one of the following: 0.1 UI/ml r-FSH (r-FSH group), 0.1 UI/ml r-LH (r-LH group), 0.1 UI/ml r-FSH and 0.1 UI/ml r-LH (r-FSH/r-LH group), 5 ␮g/ml FSH and 5 ␮g/ml LH hypophysial gonadotrophins (h-G group) as a control, or no gonadotrophins (no-G group). After in vitro fertilization with fresh ram semen, presumptive zygotes were cultured in vitro for 6–7 days and a total of 109 blastocysts were then transferred in pairs into synchronized ewes. To determine the viability of embryos after vitrification, 36 blastocysts from the r-FSH/r-LH group and 30 from the h-G group were vitrified in 10% ethylene glycol (EG) and 10% dimethylsulphoxide (DMSO) for 5 min, followed by 20% EG, 20% DMSO and 0.5 M Sucrose (S) for <45 s. They were loaded into open pulled straws (OPS) and plunged into LN2 . After warming, the blastocysts were transferred in pairs into synchronized ewes. The highest maturation rate was reached in the r-FSH/r-LH group (91.9%). However, no statistical difference was found when this group was compared with the h-G group (84.0%). Likewise, the cleavage rate of the r-FSH/r-LH group (81.4%) was not significantly different from that of the h-G group (82.3%). The cleavage rates of all other groups, however, were significantly lower than the r-FSH/r-LH and h-G groups. The blastocyst rate was highest in the h-G group (53.6%), and it was statistically higher than in the r-FSH/r-LH group (41.5%). The blastocyst rate was very similar between groups r-FSH and r-FSH/r-LH (42.0 and 41.5%, respectively). ∗ Corresponding author. Tel.: +39-079-387270; fax: +39-079-389450. E-mail address: [email protected] (C. Accardo).

0378-4320/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2003.10.004

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The lowest lambing rate (31.8%) was in the no-G group. The highest lambing rate was achieved in the r-FSH/r-LH group (66.6%). The vitrified embryos of h-G and r-FSH/r-LH groups had a very similar lambing rate (16.6% and 19.4%). In conclusion, these data provide support for the hypothesis that sheep oocytes respond to human recombinant gonadotrophins used for in vitro embryo production. © 2003 Elsevier B.V. All rights reserved. Keywords: Sheep IVM oocytes; Human recombinant gonadotrophins; Embryo development; Lambing

1. Introduction Using the in vitro technique allows us to obtain a large number of mammalian embryos for research, genetic improvement or commercial purposes. The successful culture and embryonic development of in vitro-matured oocytes has been reported in cattle, sheep and other species (Bavister et al., 1992; Tervit et al., 1995; Ptak et al., 1999; Eppig et al., 1996). However the ability to develop of mammalian oocytes matured and cultured in vitro is significantly lower than that of those produced in vivo (Dielman et al., 2002; Rizos et al., 2002). The components of the culture media and the conditions of culture can affect the meiotic regulation of mammalian oocytes (Kito and Bavister, 1997; Downs and Mastropolo, 1997). The most commonly used system is a culture medium supplemented with hormones, serum or albumin (Younis et al., 1989; Bavister et al., 1992; Thompson, 2000). The formulation of a more clearly defined medium might help to further elucidate the metabolic and nutritional requirements of mammalian oocytes and embryos produced in vitro. Moreover an improved culture media enhancing the quality of embryos may increase the cryotolerance, in turn improving the cryopreservation efficiency (Greve et al., 1993; Massip et al., 1995). Cryopreservation has seval practical applications, including embryo banking, storage of embryos during genetic testing, and economic advantages to businesses if embryos can be thawed only when and where recipients are available. Use of an optimized and standardized medium might also offer an opportunity to compare the results of different laboratories, which is often difficult. The hypophysial gonadotrophins FSH and LH and steroids play an important role in the regulation of oocyte maturation (Moor and Trouson, 1997; Younis et al., 1989; Zuelke and Brackett, 1990; Saeki et al., 1991; Downs and Mastropolo, 1997). They have been shown to enhance the in vitro fertilization and embryonic development of immature oocytes of sheep, cattle and other mammals. The commonly used commercial gonadotrophins vary in purity, and contaminants in these products may influence experimental results (Zuelke and Brackett, 1990; Choi et al., 2001). Human recombinant gonadotrophins (r-FSH and r-LH) have been demonstrated to stimulate ovulation, maturation and steroidogenesis in rats (Törnell et al., 1995). Their use for the in vitro maturation (IVM) of immature oocytes has been limited largely to studies on rodents (Törnell et al., 1995; Byskov et al., 1997; Cortvrindt et al., 1998). However, in the last few years some authors have reported the results of recombinant gonadotrophins on human and bovine embryonic development (Modina et al., 2000; Anderiesz et al., 2000; Ali and Sirard, 2002a,b).

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Serum added to the oocyte culture medium provides a source of albumin that balances the osmolality and acts as a free radical scavenger (Dunglison et al., 1995; Keskintepe et al., 1995; Downs et al., 1986; Goud et al., 1998; Thompson, 2000). However, unknown substances and contaminants may be present in sera that make it difficult to accurately define and control the contents of the culture medium (Bavister, 1995). We have used certified bovine serum albumin, which is routinely added to the in vitro maturation medium for the culture of sheep oocytes. This product minimizes variability in the maturation medium, but does not eliminate it entirely. The present study examines the effect of r-FSH, r-LH, or r-FSH/r-LH together on oocyte maturation, cleavage, embryo development and lambing rate, and compares the results to those obtained with, hypophysial gonadotrophins. The study also compares lambing rates of vitrified embryos treated with r-FSH/r-LH and h-G. 2. Materials and methods Except where otherwise indicated, all chemicals were obtained from the Sigma (St. Louis, MO, USA). 2.1. Oocytes collection Sheep ovaries were obtained from a slaughterhouse and transported to the laboratory within 2–3 h in saline solution at a temperature of approximately 35 ◦ C. Oocytes were collected by aspiration of follicles using a 20 gauge needle fitted with a 2.5 ml syringe. Follicular oocytes covered by at least two layers of granulosa cells and an evenly granulated cytoplasm was selected for in vitro maturation. 2.2. In vitro maturation Oocytes were washed in Hepes-buffered TCM199 (H-TCM199) supplemented with 0.4% BSA (bovine serum albumin fraction V, A 9418), and 2 mM glutamine. The medium used for maturation was bicarbonate-buffered TCM199 with the osmolarity adjusted to 275 mOsm and containing glutamine at a concentration of 2 mM. Oocytes were collected and divided into five different maturation groups. The basic culture medium used with all five groups consisted of TCM199 supplemented with 4 mg/ml BSA, 100 ␮M cysteamine, 0.3 mM sodium pyruvate, and 1 ␮g/ml estradiol-17␤. The no-G group was incubated in this basic medium without further additions. The culture media for the r-FSH group was supplemented with 0.1 UI/ml r-FSH (Gonal-F® 75, Serono, Italy), and the r-LH group was supplemented with 0.1 UI/ml r-LH (Lhadi® 75UI, Serono, Italy). The r-FSH/r-LH group received 0.1 UI/ml of both recombinant gonadotrophins. The h-G group was supplemented with 5 ␮g/ml FSH (Ovagen ICP, Auckland, NZ), and 5 ␮g/ml LH (from sheep pituitary). The oocytes were incubated in 400 ␮l of medium in four-well dishes (Nunc, Nunclon, Denmark) with each well containing 20–30 oocytes covered with mineral oil. All dishes were incubated at 39 ◦ C in air in a humidified atmosphere of 5% CO2 for 24 h. Following maturation, oocytes were partially stripped of surrounding cumulus cells using a fine bore pipette and 300 UI/ml

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of hyaluronidase in H-TCM199. A total of 385 oocytes (divided among the experimental groups) were fixed for 24 h in ethanol and acetic acid (3:1), stained with 2% acetorcein and observed under an inverted microscope to assess the maturation rate. Oocytes were classified as mature (MII: condensation of chromatin at the second metaphase plate) and immature (other meiotic stages). Oocytes with either clumps of chromatin or no chromatin were eliminated from the data, because the meiotic stage could not be precisely determined. 2.3. In vitro fertilization Following removal of cumulus cells the denuded oocytes were washed in fertilization medium three times. Fresh semen from a Sarda breed ram of proven fertility was collected and kept at room temperature for up to 2 h, then washed in synthetic oviduct fluid (SOF), centrifuged twice at 200 × g for 5 min and added directly to the fertilization medium. The medium used was SOF, as originally described by Tervit et al. (1972), enriched with 20% heated inactivated estrous sheep serum. A maximum of 15 oocytes per drop were fertilized in 50 ␮l with 1 × 106 sperm/ml at 39 ◦ C with 5% CO2 in humidified air for 20 h. 2.4. In vitro culture After IVF, presumptive zygotes were washed in SOF to remove spermatozoa and cellular debris. They were then allocated to 20 ␮l culture drops (five to six embryos/drop) consisting of SOF supplemented with 1% (v/v) BME-essential amino acids, 1% (v/v) MEM-nonessential amino acids, 1 mM glutamine and 8 mg/ml fatty acid free BSA. The incubation conditions were 7% O2 , 5% CO2 , and 88% N2 at 39 ◦ C in humidified air. On the third and fifth day of culture (with day 0 defined as the day of fertilization) 10% charcoal stripped foetal bovine serum (FBS) was added to the medium. The culture was continued until 6–7 days post-fertilization. 2.5. Vitrification and warming Dulbecco’s PBS supplemented with 0.3 mM Na pyruvate and 20% FBS was used as the basic vitrification solution. Blastocysts were exposed at room temperature to 10% EG and 10% DMSO for 5 min, then to 20% EG, 20% DMSO and 0.5 M Sucrose (S) for <45 s. They were loaded into open pulled straws (OPS) and immediately plunged into LN2 . The straws were warmed in a water bath at 37 ◦ C and the contents expelled into a Petri dish, then mixed in 0.5 M S for 5 min and moved into a drop of H-TCM199 and 20% FBS for the transfer to recipient ewes. 2.6. Transfer of embryos After 6–7 days of culture only 109 embryos, among those that reached expanded blastocyst stage, were transferred in pairs into recipient ewes 7 days after the onset of oestrus because of the limited number of available animals. The 66 vitrified embryos were transferred

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after warming. Pregnancy was confirmed by ultrasonography at 40 days and pregnancies were allowed to go to term. 2.7. Statistical analysis Comparison between groups was performed by the chi-square test (SAS/STAT User’s Guide, 6.03 edition, SAS Institute Inc., Cary, NC).

3. Results 3.1. In vitro maturation The lowest maturation rates were in the no-G and the r-LH groups (60.0 and 60.7%). Both were statistically different (P < 0.001) from the r-FSH and r-FSH/r-LH groups (81.9 and 91.9%, respectively). The r-FSH and r-FSH/r-LH groups were not statistically different, nor was any statistical difference found between the r-FSH/r-LH group and h-G group (91.9% versus 84.0%) (Table 1). 3.2. In vitro fertilization The cleavage rate of the no-G group was the lowest among all the groups (36.7%; P < 0.001); the highest cleavage rate (81.4%, P < 0.001) was reached when both of the recombinant hormones were added to the medium. However, when the r-FSH/r-LH group was compared with the h-G group (Table 1), no statistical difference in cleavage rate was found (81.4% versus 82.3%). 3.3. Embryo development The blastocyst rate was significantly lower in the r-FSH/r-LH group as compared to the h-G group P < 0.05 (41.5% versus 53.6%).The lowest blastocyst rate was obtained in the no-G group (12.4%), but the blastocyst rate of the r-LH group (27.0%) was also very low. Table 1 Maturation, clevage, blastocyst and lambing rate after maturation of oocytes in different media Maturation conditions

Matured oocytes: n (%)

Cleavage: n (%)

Blastocyst/cultured oocytes: n (%)

Lambs born/transferred embryos: n (%)

No gonadotrophins r-FSH r-LH r-FSH/r-LH h-G (control)

45/75 (60.0) a 59/72 (81.9) b 54/89 (60.7) a 68/74 (91.9) b 63/75 (84.0) b

64/177 (36.7) a 128/183 (69.9) b 96/174 (55.2) c 145/178 (81.4) d 149/181 (82.3) d

22/177 (12.4) a 77/183 (42.0) b 47/174 (27.0) c 74/178(41.5) b 97/181 (53.6) d

7/22 (31.8) a 11/21 (52.3) abc 10/21 (47.6) abc 16/24 (66.6) bc 13/21 (61.9) c

Values in the same column with different letters differ: (a, b) P < 0.001; (a, b; a, c; a, d; b, c; c, d) P < 0.001; (b, d) P < 0.01; (a, b; a, c; a, d; b, c; c, d) P < 0.001; (b, d) P < 0.05; (a, b) P < 0.01; (a, c) P < 0.05.

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Table 2 Lambing rate of vitrified embryos produced with recombinant or hypophisial gonadotrophins in the maturation medium of sheep oocytes Maturation conditions

Blastocyst transferred: n

Recipients: n

Lambs born/transferred embryos: n (%)

r-FSH/r-LH h-G

36 30

18 15

7/36 (19.4) 5/30 (16.6)

No significant differences were found between the r-FSH and r-FSH/LH groups (42 and 41%) (Table 1). 3.4. Lambing rate of fresh embryos The no-G group had a lambing rate (31.8%) that was significantly lower (P < 0.01) than that of the r-FSH/r-LH group (66.6%). There was no significant difference in lambing rates between the r-FSH and r-FSH/r-LH groups. Likewise, the lambing rates of the r-FSH/r-LH and h-G groups did not differ significantly (Table 1). 3.5. Lambing rate of vitrified embryos The lambing rate of ewes receiving vitrified embryos did not differ significantly between the r-FSH/r-LH and h-G groups. Out of 36 r-FSH/r-LH and 30 h-G blastocysts transferred, seven and five lambs were born, respectively, 19.4 and 16.6% (Table 2).

4. Discussion In the present study we noted that in the oocytes cultured without gonadotrophins there was no expansion of the granulosa cells. The addition of FSH and/or LH has been shown to improve the expansion of the granulosa cells in different mammalian species (Zuelke and Brackett, 1990; Singh et al., 1993; Choi et al., 2001). However, successful IVF and embryonic development occurred in all groups, including those without FSH or LH, indicating that cumulus expansion is not required for IVF and blastocyst development. This finding agrees with those of other studies (Zuelke and Brackett, 1990; Choi et al., 2001; Lonergan et al., 1996; Paula-Lopes et al., 1998; Ali and Sirard, 2002a,b). The completion of meiosis observed in this study was similar in all groups, except for the no-G and the r-LH groups. These two groups had maturation rates that were significantly lower than the other groups. LH is considered to be the gonadotrophin primarily responsible for the resumption of meiosis in vivo (Lindner et al., 1974), and some reports indicate that the addition of LH in the absence of serum enhances the meiotic maturation, fertilization and development of in vitro-matured bovine oocytes (Younis et al., 1989; Zuelke and Brackett, 1990; Saeki et al., 1991; Martins et al., 1998). Our results do no support this hypothesis, but rather are in agreement with the finding of Ali and Sirard (2002a), Anderiesz et al. (2000) and Choi et al. (2001), who demonstrated that LH alone had no effect on the developmental

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potential of bovine oocytes. This may be due to the fact that studies supporting enhanced fertilization and maturation were done with hypophysial rather than recombinant LH, where contamination with FSH and other unknown substances may have influenced the results (Zuelke and Brackett, 1990). Our data indicate that r-FSH alone or in combination with r-LH and in presence of estrogens may have stimulatory effects on the progression of the meiotic cycle. Similar results were obtained in other experiments with bovine and human oocytes (Modina et al., 2000; Anderiesz et al., 2000; Mikkelsen et al., 2000). It has been shown that gonadotrophins are clearly beneficial, even if not essential, for resumption of meiosis in in vitro cultured mammalian oocytes (Moor and Trouson, 1997; Zuelke and Brackett, 1990; Saeki et al., 1991; Eppig et al., 1996, 1998; Guler et al., 2000). The cleavage rate was statistically different in all groups, exclusive of the h-G group. Cleavage rate of the h-G group did not differ significantly from that of the r-FSH/r-LH group. The lowest cleavage rate was obtained when there were no gonadotrophins in the maturation medium. These results are very similar to those reported in cow oocytes matured in very similar conditions (Ali and Sirard, 2002a). The cleavage rate of the r-FSH group was significantly higher than that of the r-LH group. The positive effect of FSH alone, or in combination with E2 , has already been reported (Izadyar et al., 1998a,b; Ali and Sirard, 2002a). Only when the medium contained all the gonadotrophins did the cleavage rate in our study reach the highest value, which was comparable to that of the h-G group. This agrees with data previously reported for other mammalian species (Younis and Brackett, 1989; Zuelke and Brackett, 1990; Saeki et al., 1991; Kito and Bavister, 1997; Anderiesz et al., 2000; Choi et al., 2001). It is likely that steroids, gonadotrophins and the maturation medium itself all play an important role, particularly if the oocytes are matured in a defined or semi-defined culture media. This view is supported by the data of Bavister et al. (1992), Downs and Mastropolo (1997), Anderiesz et al. (2000) and Ali and Sirard (2002b), who demonstrated that the use of different culture media (M16, MEM, Ham’s F-10, TCM199, etc.), and/or minor changes in culture conditions (i.e. ±FSH, ±LH, ±EGF) can lead to a significant variation in meiotic regulation, and thus fertilization and development in mammalian oocytes. The blastocyst rate of the cultured oocytes was statistically different among the groups, except when the r-FSH and r-FSH/r-LH groups were compared. The addition of LH into the maturation medium did not increase blastocyst yield, although there have been many studies reporting the beneficial effects of LH during the IVM of oocytes (Younis et al., 1989; Zuelke and Brackett, 1993). Our data are in some ways comparable to the results of Izadyar et al. (1996), who demonstrated that when bovine oocytes were cultured in the presence of FSH and hCG, only FSH influenced IVM oocytes. It has been demonstrated by Van Tol et al. (1996) that receptors for FSH, but not for LH, are transcribed in the cumulus and granulosa cells of medium-sized (2–6 mm) follicles. This was the average size of the follicles used in our study. The lowest blastocyst rates were obtained in the no gonadotrophin group and in the r-LH group, which also had the lowest maturation and cleavage rates. The blastocyst rate in the r-FSH/r-LH group was significantly lower than that of the h-G group. It is well known that the hypophysial gonadotrophins, unlike recombinant hormones, can contain contaminants (i.e. growth factors) that can improve the blastocyst yield (Harper and Brackett, 1993; Rieger et al., 1998; Izadyar et al., 1998a,b; Lonergan et al., 1996; Guler et al., 2000).

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The highest lambing rate (66.6%) was achieved when both recombinant gonadotrophins were added to the maturation medium. However, there was no statistical difference among the groups, except when the no-G group was compared with the r-FSH/r-LH group. It may be that the low number of observations reduced the possibility of finding significant differences. Lambing rates were very similar following transfer of r-FSH/r-LH and h-G vitrified embryos. This agrees with work done previously in our laboratory (Dattena et al., 2002). It is well known that cryotolerance improves with the quality of the embryos (Fair et al., 2001; Abe et al., 1999; Crosier et al., 2000) and in vitro-produced embryos are difficult to preserve. Our current data demonstrate that, even if the cryotolerance was not improved, at least it was not adversely affected. In conclusion, these data demonstrate that sheep oocytes are responsive to recombinant gonadotrophins in vitro. Varying the relative concentrations of r-FSH and r-LH may optimize development of oocytes.

Acknowledgements We would like to thank the SERONO ITALY for providing Recombinant LH (Lhadi® 75UI). We thank Mr. G. Camoglio and Mr. A. Pintadu for expert management of the animals and Mr. F. Chessa for the assistance with experimental procedures. Part of this work was supported by Project RAIZ (RZ.287).

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