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BOR - Papers in Press, published online ahead of print February 6, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.025494
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BIOLOGY OF REPRODUCTION 70, 1634–1643 (2004)
DOI: 10.1095/biolreprod.103.025494
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Improvement of the Developmental Capacity of Oocytes from Prepubertal Cattleby Intraovarian Insulin-Like Growth Factor-I Application1

A. Oropeza3,4, C. Wrenzycki3, D. Herrmann3, K.-G. Hadeler3, and H. Niemann2,3

Department of Biotechnology,3 Institute for Animal Breeding (FAL), Mariensee, 31535 Neustadt, Germany Departamento de Producción Industrial y Animal,4 Decanato de Ciencias Veterinarias, Universidad Centroccidental "Lisandro Alvarado," Barquisimeto 3001, Venezuela


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The developmental potential of oocytes from prepubertal cattle is decreased, compared with those from their adult counterparts. The aim of the present study was to improve the developmental capacity of oocytes from prepubertal cattle by either systemic application of recombinant bovine somatotropin (rbST) or intraovarian injection of insulin-like growth factor-I (IGF-I). Blastocyst yields and the mRNA expression pattern (relative abundance, RA) of three putative marker genes (i.e., glucose transporter-1, Glut-1; eukaryotic translation initiation factor-1A, eIF1A, and upstream binding factor, UBF) were selected as criteria to determine the success of the treatments. At 6–7 mo of age, 30 healthy Holstein calves were randomly assigned to three experimental groups. The first group served as control and received an intraovarian injection of 0.6 ml acetic acid. The second group received a single s.c. injection of 500 mg of rbST. The third group received an intraovarian injection of 6 µg recombinant human IGF-I. During the following 2 wk, follicles were aspirated four times via transvaginal ultrasound-guided technology. All animals were i.m. injected with 60 mg FSH 48 h prior to each aspiration. The treatments were repeated with the same animals at 9–10, 11–12, and 14–15 mo of age. For comparison, five adult cows were each i.m. injected with 100 mg FSH and underwent oocyte retrieval. The proportion of oocytes considered to be developmentally competent was higher in cows than calves (65% vs. 58%, 50%, 52%) for the control, rbST, and IGF-I groups, respectively. The rate of blastocysts was similar in IGF-I-treated calves and cows (28% and 25%) and was higher (P <= 0.05) than in the controls and the rbST group (11% and 16%). The RA for Glut-1 was lower (P <= 0.05) in two- to four- cell embryos from calves, compared with cows. At the 8- to 16- cell stage, Glut-1 RA was similar in IGF-I-treated calves and cows. The RA for eIF1A was higher (P <= 0.05) in 8- to 16-cell embryos derived from cows than those from the control group. Results show that IGF-I intraovarian injection increased blastocyst yields and mRNA expression of Glut-1 and eIF1A to levels found in embryos produced from adult cows. This treatment may at least partially overcome the developmental deficiency of oocytes derived from calves and could be a step forward toward the use of prepubertal animals in breeding programs aimed at shortening the generation interval.

embryo, growth factors, growth hormone, oocyte development, ovum pick-up/transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Onset of puberty is characterized by changes in plasma concentration of growth hormone (GH) and changes in plasma and follicular fluid concentration of insulin-like growth factor-I (IGF-I) [1, 2]. Both molecules play an important role during follicular and embryonic development [3, 4]. Recently it has been demonstrated that increased follicular IGF-I levels via intrafollicular recombinant human (rh)IGF-I injection are associated with follicle selection in heifers [5].

The use of oocytes from calves in in vitro embryo production programs bears considerable potential for an accelerated genetic gain in domestic livestock production through a reduced generation interval [6, 7]. Ultrasound- guided ovum pick-up (OPU) via transvaginal follicular aspiration is a reliable technique for harvesting immature bovine oocytes repeatedly from live donors [8, 9]. Previously we adapted OPU for the repeated noninvasive collection of oocytes from calves and heifers [10, 11]. 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 [11–13]. Differences between oocytes from calves and cows have been found with regard to size, ultrastructure, metabolism, and cytoplasmic maturation [14–17]. With respect to metabolism, oocytes and embryos from calves show a delayed uptake of glucose and pyruvate and a reduced protein synthesis [16–18]. In early bovine embryos, glucose uptake increases markedly between 8- and 16-cell stage, coinciding with the activation of the embryonic genome, compaction, and blastulation [19–21]. Glucose is taken up into the cell by an energy- dependent mechanism (SGLT) and with the aid of facilitative glucose transporters (GLUTs 1–8) [22, 23]. IGF-I and GH affect Glut-1 mRNA expression and glucose uptake in mouse embryos [22, 24]. The mRNA for GH and IGF-I receptors have been identified in bovine cumulus cells, oocytes, and embryos [25, 26]. Supplementation of embryo culture media with GH or IGF-I improved oocyte maturation and blastocyst yields, indicating that these molecules play an important role in bovine embryonic development [25, 27, 28]. However, the effects of an IGF-I application in vivo on bovine oocyte and embryo developmental competence have not yet been studied. The gene for Glut-1 is expressed in bovine oocytes from adult donors and throughout preimplantation development. Expression is significantly affected by culture conditions [29]. It is higher in the trophectoderm than in the inner cell mass [30]. Expression of Glut-1 in embryos derived from prepubertal calves and its relationship with the developmental competence of oocytes has not been studied.

RNA and protein synthesis as well as reversible changes in the phosphorylation of specific proteins are required for germinal vesicle breakdown during bovine meiotic maturation [31–34]. It has been shown that protein synthesis is reduced in oocytes and cumulus cells from calves [18], whereas the nuclear maturation rates of oocytes from calves are similar to those of cows after 24 h of in vitro maturation [16]. Low levels of transcriptional and translational activity have been detected in zygotes and two- to four- cell stage embryos [35, 36]. However, the major activation of the bovine embryonic genome occurs at the 8- to 16-cell stage [20], which is correlated with an increased protein synthesis and changes in chromatin structure because of acetylation of core histones [33, 35, 37]. In mouse embryos, insulin and IGF-I stimulate the protein synthesis [38, 39]. Eukaryotic translation initiation factor 1A (eIF1A, formerly called eIF-4C), is a transiently expressed endogenous marker of genome activation found in mouse and bovine embryos [40, 41]. Amongst others, eIF1A is involved in the initiation of translation in eukaryotic cells [42] mainly by catalyzing the transfer of Met-tRNAfeIF2-GTP complex to the 40S ribosomal subunits to form a stable 40S preinitiation complex, thereby accomplishing 60S subunits during translation [43]. Upstream binding factor (UBF) is involved in recognizing the rRNA gene promoter and activates RNA polymerase I– mediated transcription [44]. UBF plays a critical role in synthesis and assembly of the 60S and 40S eukaryotic ribosome subunits required for protein synthesis [45]. Expression of the mRNAs for eIF1A and UBF has not been studied in embryos derived from prepubertal calves.

The objective of the present study was to determine whether a systemic GH treatment and an intraovarian IGF- I application can affect the developmental competence of oocytes from prepubertal calves. The expression pattern of three putative marker genes Glut-1, eIF1A, and UBF was assessed in in vitro-produced embryos prior to and after major genomic activation. To determine putative changes in oocyte collection efficiency and oocyte developmental competence around the onset of puberty, oocytes were retrieved over a period of 7–8 mo from the same animals starting at 6–7 mo of age. Oocytes from adult cows were included in this study to compare treatment effects.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Thirty healthy 6- to 7-mo-old (mean 190 ± 7.6 days) Holstein Friesian calves and five fertile Holstein Friesian lactating cows from the experimental herds of the Institute in Mariensee were selected for this study by careful examination of health status and development of the genital tract. Only calves with an adequate physiological status for the age of 6–7 mo were included in the present investigation. Oocyte collection was initiated at 6–7 mo of age and continued at 2-mo intervals until 14–15 mo of age to examine changes in the efficiency of oocyte collection, blastocyst yields, and the expression pattern of developmentally important gene transcripts. Oocytes were collected over two weekly periods with a total of four OPU sessions per age category by ultrasound-guided OPU at 3- to 4-day intervals. Prior to the experiment, the calves were randomly divided into three groups of 10 calves each. Calves were kept in same groups throughout the entire study, irrespective of their oocyte yields. An additional group consisted of five cows (Table 1). At each age, 5 of the 10 calves per group were used to produce 2- to 4- and 8- to 16-cell stage embryos for reverse transcription–polymerase chain reaction (RT-PCR) analysis, whereas from the other five calves blastocysts were produced. Embryos not at the appropriate stage of development were not included in this study. The cows were used to obtain oocytes as a control of development to the 2- to 4-cell, 8- to 16-cell, and blastocyst stages.


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  		TABLE 1. Overview of experimental design and treatment groups

Animals in group 1 received an intraovarian injection of 0.6 ml 0.1 M acetic acid per ovary (control) 48 h prior to each OPU session. The needle was inserted under ultrasound control into the ovarian stroma, and the fluid was injected with the aid of the OPU set-up. Group 2 received a single s.c. injection of 500 mg recombinant bovine somatotropin (rbST; Posilac; Monsanto Co., St. Louis, MO), which exerts a biological effect over a 14-day period. Group 3 received an intraovarian injection of 6 µg of rhIGF-I (R&D Systems, Wiesbaden-Nordenstadt, Germany) 48 h prior to each OPU session. Four times during the following 2 wk, all follicles >=3 mm in size were aspirated by transvaginal ultrasound–guided OPU. Forty-eight hours prior to each OPU session, all calves were injected with 60 mg FSH (Folltropin; Vetrepharm Inc., ON, Canada). The same treatment schedule was repeated with the same animal groups at the age periods of 6–7, 9–10, 11–12, and 14–15 mo of age. Group 4 included five lactating cows that were i.m. injected with 100 mg FSH. The dominant follicle was removed 4 days before the onset of the OPU sessions to ensure a homogeneous follicle population [46].

Ultrasound Equipment and Oocyte Retrieval

The ovaries were visualized employing a model CS 9000 ultrasound system (Picker, München, Germany) with a 6.5-MHz ultrasound transducer (Picker model EUP-F-331) on a 60-cm probe carrier, which consisted of a PVC rod with two tubes. The upper tube harbored a plastic rod for follicle aspiration or intraovarian injection, and the lower tube carried the cable of the ultrasound transducer. The transducer head was protected by a sanitary latex cover (Mapa GmbH, Zeven, Germany) and was inserted into the vagina. Follicles >=3 mm in diameter were aspirated for oocyte collection. OPU was accomplished with a vacuum pump (IVF Ultra Quiet; Cook Veterinary Products, Mönchengladbach, Germany) adjusted to a vacuum of 50 mm Hg (20 ml/min). Usually after aspiration of three to four follicles, oocytes were flushed from the collection needle with Dulbecco PBS medium (D5773; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) containing 1% heat-inactivated newborn calf serum (16010-159; Invitrogen, Karlsruhe, Germany), 50 IU/ml penicillin G (Sigma), 50 µg/ ml sulfate streptomycin (35500; Serva, Heidelberg, Germany), 36 µg/ml pyruvate (P3662; Sigma), 1 mg/ml D-glucose (Carl Roth GmbH, Karlsruhe, Germany), 133 µg/ml calcium chloride dihydrate (CaCl2 2 H2O; Merck, Darmstadt, Germany), and sodium heparin (2.2 IU/ml; 24590; Serva) to prevent clotting. The fluid from each animal was stored in a 50-ml conical tube; the contents thereof were immediately passed through a filter (50 µm; Jürgens, Hannover, Germany). Blood was eliminated by washing in fresh Dulbecco PBS. The contents of the filter were then washed into a 94/16-mm Petri dish and cumulus-oocyte complexes (COCs) were retrieved under a stereomicroscope at x50 magnification (Nikon, Düsseldorf, Germany). The interval between puncture and isolation of individual COCs was always kept under 30 min. The COCs were collected with a micropipette (20 µl; Brand, Wertheim, Germany) and for each animal placed into individual drops of TCM-air medium at 37°C for morphological assessment. TCM-air medium consisted of TCM 199 tissue culture medium (M2520, containing L-glutamine and 25 mM Hepes; Sigma) supplemented with 22 µg/ml pyruvate (P3662; Sigma), 350 µg/ml NaHCO3 (31437, sodium hydrogen carbonate; Riedel-de Haën AG, Seelze, Germany), 50 µg/ml gentamycin (G3632; Sigma), and 0.1% BSA–fatty acid free (A7030; Sigma).

In Vitro Maturation, Fertilization, and Culture of Bovine Oocytes and Embryos

In vitro embryo production was performed according to an established protocol [47]. COCs were classified under a stereomicroscope as follows: category I, oocytes with at least three layers of compact cumulus cells with a homogeneous evenly granulated cytoplasm; category II, oocytes with less than three layers cumulus, cytoplasm generally homogeneous; category III, oocytes with a single layer of cumulus cells, cytoplasm of irregular appearance with dark areas; category IV, completely denuded oocytes; category V, oocytes with expanded cumulus [48]. COCs classified in categories I–III were considered as suitable for in vitro embryo production (IVP). Maturation medium consisted of TCM 199 (M2520, containing l-glutamine and 25 mM Hepes; Sigma) supplemented with 22 µg/ ml pyruvate, 2.2 mg/ml NaHCO3, 50 µg/ml gentamycin, 10 IU/ml eCG, 5 IU/ml of hCG (Suigonan, Intervet, Tönisvorst, Germany), and 0.1% BSA–fatty acid free (A7030; Sigma). Oocytes were matured in a humidified atmosphere composed of 5% CO2 in air at 39°C for 24 h. Oocytes from each donor animal were matured, fertilized, and cultured in separated drops.

For fertilization in vitro, COCs were placed in Fert-TALP containing 10 µM hypotaurine (H1384; Sigma), 0.1 IU/ml heparin (24590; Serva), 1 µM epinephrine (E4250; Sigma), [HHE], and 6 mg/ml BSA (Fraction V; Sigma). Frozen semen from one bull with proven fertility for in vitro fertilization (IVF) was used throughout this experiment. For IVF, semen was prepared by a modified swim-up procedure [49, 50]. The final sperm concentration added per fertilization drop was adjusted to 1x 106 sperm/ ml. Fertilization was achieved by 18-h coincubation under the temperature and gas conditions described for maturation.

Presumptive zygotes were cultured in 30 µl of synthetic oviduct fluid supplemented with BSA–fatty acid free 4 mg/ml [51] under silicone oil (35135, Silicone DC 200 fluid; Serva) after complete removal of the adhering cumulus cells by repeated pipetting in TCM-air in atmosphere of 5% O2, 90% N2, and 5% CO2 (Air Products, Hattingen, Germany) using a modular incubation chamber (615300; ICN Biomedicals Inc., Aurora, OH) in a humidified atmosphere at 39°C. Random samples were taken from the pool of developing embryos for freezing at three different time points (40 h, 72 h, and 7–8 days). Embryos at 40 h had reached the two- to four-cell stage, whereas embryos frozen at 72 h had reached the 8- to 16-cell stage and those at 7–8 days were at the expanded blastocyst stage. Single embryos were frozen in a minimum volume (<=5 µl) of PBS plus 0.1% polyvinyl alcohol (PVA) medium in a 600 µl siliconized Eppendorf tube (no. 710136; Biozym Diagnostic GmbH, Hess Oldendorf, Germany) and stored in a –80°C freezer prior to analysis of gene expression by RT- PCR. Only embryos that had reached the appropriate developmental stages for a given time point were used in this study.

Expanded blastocysts were stained with Hoechst 33342 stain (B 2261; Sigma) 1 µl/100µl PBS containing 1mg/ml PVA to determine the cell numbers [52]. Numbers of cell nuclei (blue stained) were counted within 10 min under a fluorescence microscope 400X (BX60 F-3, Olympus Optical, Hamburg, Germany) employing a filter with 420 nm for excitation and 365 nm for emission.

Determination of the Relative Abundanceof Developmentally Important Gene Transcriptsin Bovine Embryos

For RT-PCR analysis, the relative abundances (RA) of Glut-1, eIF1A, and UBF gene transcripts from calf embryos were compared with that of cow-derived embryos. Poly (A)+ RNA was prepared from two embryos as described previously [47]. Reverse transcription (RT) was carried out in a total volume of 20 µl. Prior to RNA isolation, 1 pg of rabbit globin RNA (BRL, Gaithersburg, MD) was added as an internal standard. The RT reaction was performed in 1x buffer (50 mM KCl, 20 mM Tris-HCl, pH 8.4; Invitrogen), 5 mM MgCl2 (Invitrogen), 1 mM of each deoxynucleotide triphosphate (dNTP; Amersham, Biosciences Europe GmbH, Freiburg, Germany). Further ingredients such as 50 U murine leukemia virus reverse transcriptase, 20 U RNAse inhibitor, and 2.5 µM random hexamers were supplied by Applied Biosystems (Foster City, CA). The reaction mixture was overlaid with mineral oil (Sigma) to avoid evaporation. RT was carried out at 25°C for 10 min, followed by 1 h at 42°C, a denaturation step at 99°C for 5 min, and flash cooling at 4°C. The reaction mixture contained 0.1 cDNA embryo equivalents/µl (two embryos in a 20-µl reaction volume) and 50 fg globin RNA/µl.

PCR was performed using the cDNA equivalent of 0.5 embryos (5 µl) and 50 fg of globin RNA (1 µl) in a final volume of 50 µl of 1x buffer (50 mM KCl, 20 mM Tris-HCl, pH 8.4), 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM of each sequence-specific primer in a thermocycler (PTC- 200; MJ Research, Watertown, MA). In the case of eIF1A and UBF, a primer concentration of 1 µM was employed. To ensure specific amplification, a hot-start PCR protocol in which 1 U Taq DNA polymerase (Invitrogen) added at 72°C was employed. PCR primers were designed from the coding regions of each gene sequence using the OLIGO program. The sequences of the primers (Globin [53], Glut-1 [54], eIF1A [55]), the annealing temperatures, the fragment sizes, and sequence references are summarized in Table 2.


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  		TABLE 2. Primers used for PCR of bovine embryo gene transcript

The PCR program employed an initial denaturation step of 97°C for 2 min followed by a step of 72°C for 2 min (during which time hot-start was performed) and cycles of 15 sec at 95°C for DNA denaturation, 15 sec at the primer-specific annealing temperature, and 15 sec at 72°C for primer extension. The last cycle was followed by a 5-min extension at 72°C and cooling to 4°C (for number of cycles, see Table 2). The PCR reactions for the genes of interest and the globin internal standard were performed in separate tubes.

The RT-PCR products were subjected to electrophoresis on a 2% agarose gel in 1x TBE buffer (90 mM Tris, 90 mM borate, 2 mM EDTA, pH 8.3) containing 0.2 µg/ml ethidium bromide, present in both gel and running buffer to stabilize the concentration of ethidium bromide for quantification. The fragments were visualized on a 312-nm UV transilluminator; the image of each gel was recorded using a CCD camera (Quantix, Photometrics, München, Germany) and the IPLab Spectrum program (Signal Analytics Corp., Vienna, VA). The intensity of each band was determined by densitometry using an image analysis program (IPLab Gel). The relative amount of the mRNA of interest was calculated by dividing the intensity of the band for each developmental stage by the intensity of the corresponding globin band. For each calf group, 20 separate RT-PCR reactions with 2- to 4-cell and 8- to 16-cell stage embryos and 37 for each stage of the cow embryos were performed. For expanded blastocysts, each group was repeated in seven replicates, and each RT was performed with two embryos (each PCR reaction, each gene, required 5 µl of the RT reaction, whereas each rabbit globin PCR required only 1 µl). For each gene, a semilog plot of the fragment intensity as a function of cycle number was used to determine the range (number of cycles) over which linear amplification occurred and the number of PCR cycles was kept within this range [29]. The RNA recovery rate was estimated as the ratio between the intensity of the globin band with and without the RNA preparation procedure, starting with an equivalent of 50 fg in the PCR reaction. On average, 60% of poly (A)-tailed RNA was recovered using the Dynabead oligo d(T) mRNA isolation method, which is in agreement with previous results from our laboratory [26, 47]. This semiquantitative RT-PCR assay is highly sensitive and accurate and yields similar results as real time RT- PCR techniques (see [56]).

Statistical Analysis

The data were analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA) software package. After testing for normality using the Kolmogorov-Smirnov test with Lilliefor correction and testing of equal variance with the Levene Median test, a one-way ANOVA followed by multiple pairwise comparisons using the Tukey test was employed. Blastocyst and cleavage rates were compared between treatment groups using two- way ANOVA with the main effects being age of animals and treatment and their interactions, followed by multiple pairwise comparisons using the Tukey test. This analysis revealed no significant interactions among these factors. Differences of P <= 0.05 were considered significant.

All animal experiments were conducted in full agreement with the German animal welfare law.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Calves at 6–7 Months of Age

Results are shown in Table 3 and Figure 1. At 6–7 months of age, the number of suitable oocytes collected per group was significantly lower (P <= 0.05) for rbST-treated calves (2.7 ± 0.4) than in the control group (5.1 ± 0.6) and cows (5.8 ± 0.9). Similarly, the proportion of suitable oocytes from total oocyte yields was significantly lower (P <= 0.05) in rbST-treated calves (39%) and IGF-I-treated calves (45%) than in the cows (62%). The cleavage rate (% 2 cells) was lower (P <= 0.05) in all groups of calves than for adult cows. The blastocyst rate was similar for all groups of calves. However, from adult cows it was significantly (P <= 0.05) higher than in all groups of calves (Table 3). The RA of Glut-1 in two- to four-cell embryos derived from calves was lower (P <= 0.05) than in those derived from cows (Fig. 2), whereas in 8- to 16-cell embryos from the controls and rbST-treated calves the RA was significantly lower (P <= 0.05) than in embryos from cows. Only for embryos from the control group was the RA for eIF1A mRNA lower (P <= 0.05) than in cows (Fig. 2). Within the control group, differences (P <= 0.05) in the RA of Glut-1 for two- to four-cell embryos were detected between 6–7 months and the other age categories (Fig. 2).


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  		TABLE 3. Developmental competence of oocytes collected from calves at 6–7 months of age



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  		FIG. 1. Averages of suitable oocytes, cleavage rate, and blastocyst rate in calves at different ages compared with adult cows. Suitable oocytes, bars with different superscript within age-matched (6–7 mo) and total differ significantly (a, b, c, P <= 0.05), ANOVA one way. Bars with different superscript between age-matched and adult cows differ significantly (A, B, P <= 0.05), ANOVA one way. Two-cell embryos, bars with different superscript between age-matched and adult cows differ significantly (a, b, P <= 0.05), ANOVA two way. Bars with different superscripts within groups (A, B) and total (a, b) differ significantly (P <= 0.05), ANOVA two way. Blastocysts, bar with different superscripts within age-matched and compared with adult cows differ significantly (a, b, c, P <= 0.05), ANOVA two way. Bar with different superscript within group (A, B) and total (a, b) differ significantly (P <= 0.05), ANOVA two way



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  		FIG. 2. Expression pattern of Glut-1 and eIF1A in 2- to 4 and 8- to 16- cell embryo stages originating from calf oocytes of the three treatment groups, compared with embryos originating from oocytes derived from cows. Bars with different superscripts within each age-matched and in comparison with the bar from cows differ significantly (a, b, P <= 0.05). Bar with different superscripts between age-matched differ significantly (A, B, P <= 0.05)

Four of the 10 calves in the rbST group did not respond to the FSH treatment, and no developmentally competent oocytes were collected.

Calves at 9–10 Months of Age

Results are shown in Table 4 and Figure 1. At 9–10 months of age, the number of suitable oocytes was similar in all groups of calves. It was significantly lower for rbST- treated calves (2.9 ± 0.4) than in cows (6.8 ± 1.7). The proportion of competent oocytes of the total yields was significantly lower in rbST- (47%) and IGF-I-treated calves (50%), compared with cows (72%). Although the majority of the calves (90%; n = 27) were still prepubertal and showed no evidence of ovulation, cleavage rates were similar to those for oocytes from cows (60%, 74%, 70%, and 80% for the control, rbST, and IGF-I groups and cows, respectively). The blastocyst rate was similar for all groups (Table 4). The RA of Glut-1 was significantly lower (P <= 0.05) in two- to four-cell embryos derived from oocytes collected in the rbST and IGF-I groups than in cows (Fig. 2). For 8- to 16-cell embryos, the Glut-1 mRNA level was lower (P <= 0.05) only for the rbST group, compared with their counterparts from adult cows (Fig. 2).


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  		TABLE 4. Developmental competence of oocytes collected from calves at 9–10 months of age

Calves at 11–12 Months of Age

Results are shown in Table 5 and Figure 1. At 11–12 months of age, a significantly lower number of suitable oocytes could be collected from the heifers in the controls and rbST group, compared with cows. The cleavage rate was similar for oocytes derived from calves and cows. The blastocyst rate was higher (P <= 0.05) in the IGF-I group than in all other groups of calves but similar to the cows (Table 5). The RA of Glut-1 in two- to four-cell embryos was lower (P <= 0.05) for embryos from the rbST group than in cows, whereas it was significantly lower (P <= 0.05) in 8- to 16-cell embryos derived from the control group than for embryos from adult cows and IGF-I-treated calves (Fig. 2).


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  		TABLE 5. Developmental competence of oocytes collected from calves at 11–12 months of age

Calves at 14–15 Months of Age

Results are shown in Table 6 and Figure 1. At 14–15 months of age, the number of competent oocytes was lower (2.1 ± 0.3 and 2.9 ± 0.3; P <= 0.05) for the rbST and IGF- I calf groups, respectively, than in cows (4.7 ± 0.6). However, the rates of cleavage and blastocyst development were similar in all treatment groups (Table 6). The RA of Glut- 1 in two- to four-cell embryos was lower (P <= 0.05) for the rbST group than in cows. In 8- to 16-cell embryos, the RA did not differ for any of the gene transcripts tested (Fig. 2). The majority of the heifers (97%, n = 29) had shown evidence of ovulation determined by ultrasound visualization of a corpus luteum.


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  		TABLE 6. Developmental competence of oocytes collected from calves at 14–15 months of age

General Aspects of Oocytes Development among Calves and Cows

Figure 3 shows representative gels from the semiquantitative RT-PCR analysis of Glut-1, eIF1A, and UBF transcripts and the corresponding rabbit globin bands in 2- to 4- and 8- to 16-cell stage embryos derived from calves in the three treatment groups, compared in each case with their counterparts derived from cows. The effects of the different treatments (i.e., rbST and IGF-I applications) on the RA of Glut-1 and eIF1A gene transcripts in 2- to 4- and 8- to 16-cell embryos derived from calves at different age categories and adult cows are shown in Figure 2. No significant differences were detected for UBF expression in 2- to 4- and 8- to 16-cell embryos and for eIF1A in 2- to 4-cell embryos (not shown). No significant differences were detected in expanded blastocyst for any of the three gene transcripts tested (not shown). Expanded blastocysts from calves and cows contained a similar number of cells, i.e., 102 ± 8 controls, 108 ± 9 rbST, 109 ± 10 IGF-I, and 120 ± 4 for cow blastocysts (9 blastocysts per group). A comparison of the pooled results demonstrated that fewer (P <= 0.05) suitable oocytes were collected from calves irrespective of treatment than from cows and that the cleavage rate was significantly lower (P <= 0.05) for oocytes retrieved from calves than those from adult cows (Table 7 and Fig. 1). In contrast, calves treated with IGF-I produced oocytes that yielded a blastocyst rate that was similar to that of oocytes obtained from cows (Table 7 and Fig. 1). Pooling the various age categories of calf groups also demonstrated that two- to four-cell embryos had a significantly lower (P <= 0.05) RA for Glut-1 than their adult counterparts. The RA for Glut-1 in 8- to 16-cell embryos from control and rbST groups was significantly lower (P <= 0.05) than in embryos from adult cows. The RA for eIF1A was significantly lower (P <= 0.05) only for embryos from the control calves, compared with those from cows (Fig. 2). The RA for the IGF-I group in 8- to 16-cell embryos was similar to the RA for cows in the three gene transcripts.



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  		FIG. 3. Representative photograph of gels from semiquantitative RT-PCR analysis for the three transcripts analyzed in this study. Photographs of representative gels produced from semiquantitative RT-PCR analysis of Glut-1, UBF and eIF1A transcripts and the rabbit globin standard in 2- to 4- and 8- to 16-cell embryo stages originating from calf oocytes from the three treatment groups (control, rbST, and IGF-I) in comparison with embryos originating from oocytes derived from cows


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  		TABLE 7. Overall developmental competence of oocytes collected from calves 6–15 months of age


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study is the first to investigate the effects of systemic somatotropin and 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 treatment. Results of this study add novel information on calf oocyte development and show ways to improve the reduced developmental competence of calf oocytes.

Recombinant human IGF-I was chosen for this study because it has an identical amino acid composition with the bovine IGF-I [57]. Previously, it was shown that Glut-1 mRNA expression was affected in mice oocytes by IGF-I [24]. Furthermore, glucose uptake by the blastocyst was increased in the presence of IGF-I and insulin through the type 1 IGF receptor [58], 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 [4, 59, 60] and was correlated with an increased number of follicles <5 mm of diameter [61]. In the present study, 4 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 therefore had the lowest average of suitable oocytes. Previously it had been reported that approximately 30% of the prepubertal donors did not respond to a treatment with FSH [62].

Fewer competent oocytes were recovered from calves than from cows, and treatment with rbST or IGF-I did not increase at any age category the number of competent oocytes, compared with the control calves. This is in agreement with a previous study employing 15-mo-old heifers in which application of rbST increased the number of follicles, whereas the number of competent oocytes remained low [63]. 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 [64]. The proportion of competent oocytes at 6–7 months of age in the present study coincides with previous reports, in which 37% to 39% of the oocytes recovered from superovulated calves were considered suitable for IVP [62, 65]. On the contrary, others have reported >90% competent oocytes to be collected from 7-mo-old calves after stimulation with FSH [12]. The proportion of competent oocytes obtained in the present study from 9- to 10-mo-old calves (47% to 61%) coincides with the results reported previously for 9- to 10-mo-old animals (51%) [12], indicating individual variability among donors [66]. This could also explain the low proportion of competent 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 been reported. The proportion of competent oocytes in the calves after reaching puberty was similar to that of cows, which is consistent with previous reports [12, 67].

Both the rbST and IGF-I treatments were unable to improve cleavage rates in oocytes from calves at 6–7 months of age. The age markedly affected cleavage rates (37% to 45%) in oocytes from 6- to 7-mo-old calves, and the treatments did not lead to improvements. Similar cleavage rates were previously reported [12, 68]. However, also a cleavage rate of 73% was achieved after fertilization of oocytes from abattoir ovaries of stimulated 3-mo-old calves that were matured on a monolayer of granulosa cells with 10% fetal calf serum [69]. In agreement with a recent report, in the present study, cleavage rates were similar in calves from the age of 9–10 months onward and cows [12]. Beneficial effects of an rbST treatment on embryonic development in superovulated cows and in in vitro cultured oocytes have been reported [70, 71].

The initial period of mammalian preimplantation development prior to the major embryonic genomic activation is under the control of maternal transcripts and polypeptide molecules synthesized during oocyte growth and accumulated during late stages of follicular growth [72]. In the present study, neither treatment with somatotropin nor IGF- I increased Glut-1 expression in 2- to 4-cell embryos from calves indicating that the age of the donor is crucial for the regulation of transcription level. Differences in protein synthesis between calf and cow oocytes and granulosa cells and in the glucose uptake in two- to four-cell embryos were reported previously [13, 18, 73] 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, indicating that prepubertal oocytes are unable to respond to factors present in the follicular fluid and that the specific receptors appear around puberty [74]. The presence of mRNA type 1 IGF receptors has been detected in granulosa cells and oocytes from preantral and antral follicles from adult cows [75, 76]. 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, oocytes, or both.

The low protein synthesis observed in calf oocytes [18] is possibly mediated by factors other than upstream binding factor and eukaryotic initiation factor 1A because no differences in these gene transcripts were detected between two- and four-cell embryos derived from the different treatment groups.

Low transcriptional activity has been observed already in bovine 2-cell embryos [36]; the major activation of the genome, associated with important changes in protein synthesis, occurs at 8- to 16-cell stage [20]. Previously, this was a critical step during in vitro embryo culture and was frequently associated with an early developmental arrest [77, 78]. In the present study, embryos at the 8- to 16-cell stage derived from IGF-I-treated calves had an mRNA abundance for Glut-1 and eIF1A 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 during the activation of the genome. This prepares the embryo for an efficient protein synthesis and metabolism of glucose [39, 58]. A positive correlation had been suggested between mRNA contents during early developmental stages and the developmental competence of bovine oocytes [79]. 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 2-cell embryos progressing to the blastocyst stage. Glucose is a critical energy source during compaction and blastulation in bovine embryos [80], and its uptake is significantly increased from the 16-cell stage to the blastocyst [13, 21]. In the present study, we found a low mRNA abundance for Glut-1 in 8- to 16-cell calf embryos in the control and rbST groups, compared with their adult counterparts. Previously, differences in glucose uptake between calf and cow embryos from the 8- to 16-cell stage were not observed [13]. 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 [23]. In cattle and mice, it has been shown that Glut-1 is located in a basolateral position in the cells, and in the mouse Glut-1 plays a fundamental role in the transport of glucose from the trophectoderm to the cells of the inner cell mass [22, 23]. A similar mechanism in bovine embryos could explain the lower blastocyst rates obtained in the control and rbST groups of 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. Our results are consistent with previous reports in which a positive effect of IGF-I was observed on the blastocyst rate in bovine embryo culture [27, 28, 81].

In conclusion, the present results demonstrate for the first time that IGF-I affects expression of Glut-1 and suggests that in turn the transport of glucose into the cells during compaction and blastulation is stimulated and development of blastocysts is improved. The higher RA for eIF1A is probably a mechanism whereby IGF-I increases protein synthesis in embryos. These results demonstrate that the reduced developmental competence of oocytes from prepubertal calves is attributed to a deficient expression of facilitative glucose transporters and insufficient protein translation. This deficiency can at least partially be overcome by intraovarian IGF-I application, in particular when the donors are close to reaching puberty.


   ACKNOWLEDGMENTS
 
The authors are grateful for the support of the Monsanto Co., St. Louis, Missouri, for providing Posilac. The authors are grateful to H.-G. Sander, R. Poppenga, G. Möller, K. Korsawe, and S. Ponebsek for their continuous skilled assistance throughout this study. This report is dedicated to Adriana and Daniela.


   FOOTNOTES
 
1 Supported by the German Academic Exchange Service (DAAD) and Fudayacucho (Fellowship for A.O.). Back

2 Correspondence: Heiner Niemann, Department of Biotechnology, Institute for Animal Breeding (FAL), Mariensee, 31535 Neustadt, Germany. FAX: 00 49 5034 871101; niemann{at}tzv.fal.de Back

Received: 14 November 2003.

First decision: 14 December 2003.

Accepted: 22 January 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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