DISCUSSION - Improvement of the developmental capacity of oocytes from prepubertal cattle by in

Previously several attempts have been made to increase the developmental competence of oocytes derived from prepubertal calves. Hormonal treatments with FSH, eCG, and steroids (estradiol valerate and intravaginal progestagen releasing sponges), and their combinations prior to follicle aspiration have shown favorable effects on the number of aspirated follicles, number of total and competent oocytes and the development rate of these oocytes to the blastocyst stage (ARMSTRONG et al. 1994; GALLI et al. 2001; KUWER et al. 1999; PRESICCE et al. 1997). Different maturation and culture systems have been also tested. Maturation medium supplemented with hormones (FSH, 17β estradiol, hCG), fetal calf serum (FCS) or estrous cow serum (ECS) and culture medium supplemented with BSA or a culture system using a bovine granulosa cells monolayer have also contributed to improve the developmental competence of oocytes derived from prepubertal calves (KUWER et al. 1999; PRESICCE et al. 1997; REVEL et al. 1995). However, despite progress in the past few years, the rate of viable blastocysts derived from prepubertal donors is low in comparison with their adult counterparts.

The present study is the first to investigate the effects of a systemic somatotropin and local intraovarian IGF-I treatment on developmental competence of calf oocytes and mRNA expression of developmentally important genes in preimplantation bovine embryos derived thereof. Differences in the developmental competence of oocytes and the mRNA expression are likely related to the treatments. Results of this study add novel information to calf oocyte development and show ways to improve the reduced developmental competence of calf oocytes.

As described by WRENZYCKI et al. (1999), the changes in mRNA expression detected in this study reflect real differences in the amount of the transcripts present at a certain point of development since similar embryo cDNA equivalents from calves and adult cows were subjected to PCR. This rules out the possibility that the observed differences were attributed to variation in PCR conditions. For all primer pairs the number of PCR cycles was kept within the linear range of amplification. The CV levels were only marginally different and consistent with previous studies from

Discussion

this laboratory (WRENZYCKI et al. 1999; YASEEN 2002), which indicates that the semi-quantitative RT-PCR assay is reliable and highly reproducible. However, it should be taken into account that due to the possible variation in efficiency in the reverse transcription reaction and the different amplification efficiencies of each PCR reaction, the results must be viewed as relative and the level of one gene transcript should not be compared with that of a different transcript (TEMELES et al. 1994;

WRENZYCKI et al. 1999). This is also true for other techniques such as Real-time RT-PCR, or Northern blotting (BUSTIN 2000; BUSTIN 2002; PARKER and BARNES 1999). In all cases, it is possible to improve quantification by calibration of the amplification system using known amounts of an artificial gene construct which contains the correct template for the amplicon and the primer sites but even in these cases the estimation of copy number is highly dependent on the quality of the calibration. Another approach is to prepare an artificial template with a small alteration which permits the resultant amplicon to be distinguished from the native amplicon by restriction enzyme cutting. An aliquot of the calibration solution can be added to the mRNA stock prior to RT and after the PCR step and restriction enzyme digestion, the two products are compared. This so called “competitive PCR” is not without its own artifacts and is in most cases not practical. The semi-quantitative RT-PCR assay used in these experiments is highly sensitive and highly accurate and yields results which are similar to those obtained by Real Time RT-PCR technique (see KNIJN et al. 2002).

Previous studies from this laboratory (WRENZYCKI et al. 1996; WRENZYCKI et al.

1999) demonstrated that after optimization, the RT-PCR assay was sensitive enough to detect specific bovine mRNA from tongue epithelium at levels from 0.5 – 5 ng and from 0.5 blastocyst equivalents. In addition, semi-quantitative RT-PCR has been successfully employed to detect differences between specific mRNA transcripts derived from single blastocysts with similar cell numbers (WRENZYCKI et al. 2002;

WRENZYCKI et al. 2003). In the present study, two embryos of a given group were pooled for the RT reaction and 0.5 embryo equivalents (1/4 if the resultant cDNA product) were used for each PCR reaction (i.e. for each gene studied). This assay was satisfactory for the determination of differences between mRNA transcripts in

Discussion

embryos derived from different age-matched calves and cows as well as differences between treatment groups.

Previously, it was shown that Glut-1 mRNA expression was affected in mice oocytes by IGF-I (ZHOU et al. 2000). Furthermore, glucose uptake by the blastocyst was increased in the presence of IGF-I and insulin through the type 1 IGF receptor (PANTALEON and KAYE 1996) suggesting a metabolic role in the ovarian follicle. It has also been demonstrated that treatment with rbST led to increased IGF-I levels in plasma and follicular fluid (GONG et al. 1993c; GRAF et al. 1991; HERRLER et al.

1994) and was correlated with an increased number of follicles < 5 mm of diameter (GONG et al. 1997).

In the present study, 4 out of 10 calves in the rbST group did not respond to the treatment with FSH and no suitable oocytes were recovered from these animals. This group had therefore the lowest average of aspirated follicles, total oocytes and suitable oocytes. Previously, it had been reported that approximately 30% of the prepubertal donors did not respond to a treatment with FSH (FRY et al. 1998).

The proportions of recovered oocytes with respect to the number of aspirated follicles were similar for all treatment groups and all ages ruling out the possibility that the differences between the groups can be attributed to this factor.

Overall, fewer follicles were aspirated and fewer total and suitable oocytes were obtained from calves than from cows. Treatments with either rbST or IGF-I did not increase the number of follicles at any age category and the number of total and suitable oocytes compared to the control calves. In a previous study employing 15 months old heifers the application of rbST increased the number of follicles while the number of suitable oocytes remained low (BOLS et al. 1998). In contrast, heifers and cows injected once with rbST produced more high-quality oocytes but no differences in follicle numbers were observed (PAVLOK et al. 1996). Similarly, the number of follicles in adult Holstein cows was not altered by infusion of IGF-I via an implanted osmotic minipump into the ovarian stroma for 7 d beginning the day after ovulation (SPICER et al. 2000). Most studies using rbST in cattle report that the effect of this hormone is an increase in the number of small follicles (<5 mm) associated with an increase of IGF-I levels but that there is no effect on the number of medium and large

Discussion

follicles (BURATINI, JR. et al. 2000; GONG et al. 1993a; GONG et al. 1993b;

PAVLOK et al. 1996). In the present study, only the aspirated follicles were counted, the majority of which were medium or large in size. Small follicles were difficult or impossible to count. Possibly this is the reason why an increase in the number of follicles was not observed in the rbST treated animals.

The proportion of suitable oocytes at 6-7 months of age in the present study coincides with previous reports, in which 37-39% of the oocytes recovered from superovulated calves were considered suitable for IVP (BROGLIATTI et al. 1997;

FRY et al. 1998). On the contrary, others have reported >90% suitable oocytes to be collected from 7 months old calves after stimulation with FSH (PRESICCE et al.

1997). The proportion of suitable oocytes obtained in the present study from 9-10 months old calves (47-61%) coincides with the results reported previously for 9-10 months old animals (51%) (PRESICCE et al. 1997) indicating individual variability among donors (FRY et al. 1998; MERMILLOD et al. 1997; OROPEZA and BASTIDAS 2003). This could also explain the low proportion of suitable oocytes obtained in the rbST and IGF-I groups at 6-7 and 9-10 months of age compared with the cows. A detrimental effect of rbST and IGF-I on ovarian activity has not yet been reported. The proportion of suitable oocytes in the calves after reaching puberty was similar to that of cows which is consistent with previous reports (BUNGARTZ et al.

1995; PRESICCE et al. 1997; REIS et al. 2002).

In the present study, a decline in the number of aspirated follicles and collected oocytes was observed within all calf groups after repeated follicle aspirations (Fig.

39), whereas the number of suitable oocytes remained similar across all OPU sessions, with the only exception of the control group. This is consistent with previous studies in prepubertal and matured heifers in which repeated transvaginal follicle aspirations were associated with a reduced number of aspirated follicles and collected oocytes without affecting the number of suitable oocytes (MAJERUS et al.

1999; REIS et al. 2002). In contrast, in the present study, the number of aspirated follicles, collected and suitable oocytes was not affected in the cows by repeated collections. Similar results were obtained by STUBBINGS et al. (1990); STUBBINGS and WALTON (1995). However, HASLER (1998) observed, a linear decrease in

Discussion

numbers of collected oocyte relative to number of OPU sessions in 20 OPU collections per cow.

The age markedly affected cleavage rates (37% - 45%) in oocytes from 6-7 months old calves and the treatments either rbST or IGF-I did not lead to improvements.

Similar observations have been reported for prepubertal calf oocytes compared to cow oocytes (PALMA et al. 1993; LOONEY et al. 1995; PRESICCE et al. 1997;

STEEVES et al. 1999). The cleavage rates obtained in the present study for oocytes derived from calves 6-7 months of age were higher than those (21-24%) for oocytes derived from 5.3 months old calves reported by FRY et al. (1998) and similar to those (49%, 41%, 43% and 36.7%) previously reported by PRESICCE et al. (1997), TANEJA et al. (2000) and CHOHAN and HUNTER (2004) for oocytes derived from 7, 2-3, 4-5 months old calves and 7.5 months-to-term bovine fetuses, respectively.

However, also cleavage rates of 73-81% have been achieved after fertilization of oocytes, derived from the ovaries of stimulated and unstimulated 3 months old calves after slaughter, that were matured on a monolayer of granulosa cells with 10% FCS (REVEL et al. 1995). In agreement with previous reports, in the present study cleavage rates were similar in calves from the age of 9-10 months onwards and cows (LOONEY et al. 1995; PRESICCE et al. 1997). The cleavage and blastocyst rates obtained in the present study for oocytes derived from adult cows coincide with those previously reported (LOONEY et al. 1995; PRESICCE et al. 1997; REVEL et al.

1995).

Beneficial effects of an rbST treatment on embryonic development in in vitro cultured oocytes and superovulated cows have been reported (IZADYAR et al. 1996;

MOREIRA et al. 2002a). Similarly, an increase the cleavage rate has been observed in bovine and equine cumulus oocytes complexes matured in medium supplemented with IGF-I and EGF or IGF-I and gonadotropins (CARNEIRO et al. 2001; RIEGER et al. 1998). Nevertheless, HASLER et al. (2003) did not observe beneficial effects neither in the response to superovulation in Angus cows treated with rbST nor in the pregnancy rate in beef and dairy embryo recipient heifers treated with rbST.

The initial period of mammalian preimplantation development prior to the major embryonic genomic activation is under the control of maternal transcripts and

Discussion

polypeptide molecules synthesized during oocyte growth and accumulated during the late stages of follicular growth (SCHULTZ 1993). In the present study, neither treatment with somatotropin nor IGF-I increased Glut-1 expression in 2-4 cells embryos from calves indicating that the age of the donor is crucial for the regulation of transcriptional level. Previously, differences in protein synthesis between calf and cow oocytes and granulosa cells and in the glucose uptake in 2-4-cell embryos were reported (GANDOLFI et al. 1998; KHATIR et al. 1998a; STEEVES et al. 1999), and are consistent with the results reported here. Supplementation of media for calf oocytes with cow follicular fluid during in vitro maturation did not improve their developmental competence. This suggests that prepubertal oocytes are unable to respond to factors present in the follicular fluid and that the specific receptors first appear around puberty (KHATIR et al. 1997). The presence of mRNA type 1 IGF receptors has been detected in granulosa cells and oocytes from preantral and antral follicles from adult cows (ARMSTRONG et al. 2000; ARMSTRONG et al. 2002). In agreement with this observation, in the present study the RA of Glut-1 showed a tendency to increase with the age of the donor likely mediated by more type 1 IGF-I receptors in the granulosa cells and/or oocytes.

The low protein synthesis observed in calf oocytes (GANDOLFI et al. 1998), is possibly mediated by other factors than upstream binding factor and eukaryotic initiation factor 1A because no differences in these gene transcripts were detected between 2-4-cell embryos derived from the different treatment groups.

Low transcriptional activity has been observed already in bovine 2-cell embryos (VIUFF et al. 1996); the major activation of the genome, associated with important changes in protein synthesis, occurs at 8-16 cells stage (TELFORD et al. 1990).

Previously, this was a critical step during in vitro embryo culture and was frequently associated with an early developmental arrest (EYESTONE and FIRST 1991). In the present study, embryos at the 8-16 cell stage derived from IGF-I treated calves had an mRNA abundance for Glut-1 and eIF1A that was similar to their adult counterparts, suggesting that the treatment with IGF-I changed conditions in the follicle favorably to stimulate transcription of Glut-1 and eIF1A mRNA during the activation of the genome. This prepares the embryo for an efficient protein synthesis

Discussion

and metabolism of glucose (HARVEY and KAYE 1991; PANTALEON and KAYE 1996). A positive correlation had been suggested between mRNA contents during early developmental stages and the developmental competence for bovine oocytes (LEQUARRE et al. 1997). Moreover, the transport of glucose in embryos is stimulated by IGF-I and insulin. This effect is mediated by the IGF-I receptor (PANTALEON and KAYE 1996). In the present study, the higher mRNA expression for Glut-1 and eIF1A in embryos derived from IGF-I treated calves and cows was associated with a higher proportion (42 and 33% for IGF-I calves and cows vs. 20 and 21% for control and rbST groups) of two-cell embryos progressing to the blastocyst stage. Glucose is a critical energy source during compaction and blastulation in bovine embryos (THOMPSON et al. 2000), and its uptake is significantly increased from the 16-cell stage to the blastocyst stage (STEEVES et al.

1999; KHURANA and NIEMANN 2000). In addition, expression of Glut-1 provides a coordinating link with the proliferative and morphological effects of growth factors such as GH and IGF-I (IZADYAR et al. 1996; SIRISATHIEN et al. 2003). In the present study a low mRNA abundance for Glut-1 was found in 8-16 calf embryos in the control and rbST groups compared with their adult counterparts. Previously differences in the glucose uptake between calf and cow embryos from 8-16-cell stage were not observed (STEEVES et al. 1999). These results suggest that glucose uptake from the medium is not a critical step during incorporation into the cell and probably involves other glucose transporters such as Glut-3 and Glut-8 (AUGUSTIN et al. 2001). In the mouse, Glut-3 is apically distributed on the outer cells of the compacted morula and the trophectodermal cells of the blastocysts and is responsible for the uptake of glucose from the external medium while Glut-1 is located in a basolateral position of the trophectodermal cells and is responsible for the delivery of glucose to the blastocoel (PANTALEON et al. 1997a). Glut-1 is evenly distributed in the inner cell mass (ICM) cells and plays a fundamental role in the uptake of glucose by these cells (PANTALEON and KAYE 1998). In cattle, Glut-1 has been also identified in a basolateral position in the trophectodermal cells and is heterogeneously distributed in the ICM of the blastocyst (AUGUSTIN et al. 2001).

These observations allow proposing that a similar mechanism of glucose transporter

Discussion

exist in bovine embryos as in mice embryos. The low mRNA expression of Glut-1 determined in 8-16-cell embryo stages derived from control and rbST groups could compromise the transport of glucose among the blastomeres and from the blastocoel during compaction and blastulation of these embryos. This may explain the lower blastocyst rates obtained in the control and rbST groups in this study. In contrast, the RA for Glut-1 in embryos from IGF-I treated calves was similar to that from cows coinciding with a higher percentage of oocytes reaching the blastocyst stage. These results are consistent with previous reports in which a positive effect of IGF-I was observed on the blastocyst rate in bovine in vitro embryo culture (HERRLER et al.

1992; PRELLE et al. 2001; SIRISATHIEN et al. 2003). Intercellular communication between blastomeres has been observed in bovine embryos from the morula stage onwards (BONI et al. 1999). Using oocytes derived from rbST and IGF-I groups, the proportion of embryos which developed to the blastocyst stage tended to increase with the age of the donor animal, which suggests an increase in the number of receptors for GH and IGF-I in these oocytes and/or follicles. Messenger RNAs for GH and IGF-I receptors have been identified in bovine cumulus cells, oocytes and embryos (IZADYAR et al. 1997a; YASEEN et al. 2001).

The concentration of mRNA in cultured bovine embryos declines from the mature oocyte to the 8-16-cell stage and increases again at the blastocyst stage (BILODEAU-GOESEELS and SCHULTZ 1997b). The expression level of Glut-1 observed in the control calf and the mature cow groups in this study seem to fit this general pattern. However, the similar RA for Glut-1 found for 2-4-cell and 8-16-cell embryos in the rbST and IGF-I groups suggests that there was some stabilizing effect on mRNA levels caused by these treatments. This interpretation could be consistent with the results of other studies in which a constant expression pattern for Glut-1 in early embryos from the 2-cell to 8-cell stage was observed (LEQUARRE et al. 1997;

WRENZYCKI et al. 1999).

In all groups, the RA for Glut-1 in blastocysts was higher than that observed in all other embryonic stages confirming previous studies in which a higher RA was reported for Glut-1 mRNA in blastocysts than in 2-4-cell embryos and 8-16-cell embryos (WRENZYCKI et al. 1999). Similarly, in the mouse the level of the Glut-1

Discussion

protein increased 20-fold from the unfertilized oocyte to the blastocyst stage (MORITA et al. 1992).

In a previous study, a transient increase of the relative abundance of eIF1A mRNA was detected only in 8-cell embryos and it was associated with the activation of the bovine embryonic genome (DE SOUSA et al. 1998b). In contrast, in the present study the mRNA transcript for eIF1A was detected in all embryonic stages analyzed (i.e. 2-4-cell, 8-16-cell and blastocyst stages). A steady increase in the RA for eIF1A from 2-4-cell embryos to blastocysts in rbST and cows groups was observed, which is consistent with previous findings in the mouse (DE SOUSA et al. 1998b). This expression pattern was also observed in embryos derived from IGF-I treated calves, although differences were noted only when blastocysts were compared with early (2-4 cell and 8-16 cell) embryonic stages (Fig. 37). In contrast, in the control group the expression pattern for eIF1A tended to be lower in 8-16-cell embryos than in 2-4-cell embryos. This could be an important aspect for further studies because a delayed transcription of this factor could be one reason for the low blastocyst rate obtained in the control group. The pattern of mRNA loss and re-accumulation observed in bovine embryos is similar to the pattern observed in mice, although the increase occurs at the 8-cell stage in mice, possibly as a result of the earlier onset of embryonic gene activation in this species (PIKO and CLEGG 1982). Possibly, the increase in the RA

Im Dokument Improvement of the developmental capacity of oocytes from prepubertal cattle by intraovarian IGF-I application (Seite 131-141)