Developmental differences of oocytes derived from prepubertal calves

As in most mammalian species, in cattle folliculogenesis occurs during fetal development. Antral follicles are observed in fetal ovaries during late gestation and as many as 50 antral follicles appear when a calf reaches 2 months of age

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programs bears considerable potential for an accelerated genetic gain in domestic livestock production through a reduced generation interval (LOHUIS 1995; DUBY et al. 1996; ARMSTRONG et al. 1997). 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 (PRESICCE et al. 1997; STEEVES et al.

1999).

Most of the studies have reported that oocytes from young calves are deficient in their ability to develop to blastocysts and to produce successful pregnancies after embryo transfer in comparison with embryos derived from adult animal oocytes (PALMA et al. 1993; LOONEY et al. 1995; RICK 1996; KHATIR et al. 1998b). The rate of production for blastocysts was of 9-11% in nonstimulated and stimulated calves compared with 20% in adult cows (REVEL et al. 1995). Similarly, DAMIANI et al. (1996) found a low (6%) development rate to the blastocyst stage in oocytes from prepubertal calves in comparison with 33% for oocytes from adult cows. PRESICCE et al. (1997) showed that the developmental competence of oocytes from prepubertal calves is acquired with age. Oocytes from stimulated calves at 5 and 7 months of age had a lower rate of cleavage (24 and 49%) and development to blastocysts (0 and 17% / cleaved oocytes) than oocytes from adult cows (62% and 27%) for cleavage and blastocyst rate, respectively. However, when at 9 and 11 months of age these same animals (now pubertal heifers) were subjected to oocyte retrieval by ultrasound-guided ovum pick up (OPU) the rate of cleavage was not different to that of adult cows. Similarly, LOONEY et al. (1995) obtained a lower cleavage rate (33.8%) and a lower blastocyst rate (0%) for oocytes derived from prepubertal calves than for oocytes derived from the same heifers after they had reached puberty (63.4% and 18.9%) and for those from adult cows (73.3% and 31.6% for cleavage and blastocyst rate, respectively). In addition, STEEVES et al. (1999), determined that development to blastocysts of oocytes from 5 to 7 months old calves, was lower (9.8%) than of oocytes from adult cows (33.7%). In a recent study, CHOHAN and HUNTER (2004) observed a lower maturation rate (80.1% vs. 92.0%), fertilization rate (69.3% vs. 79.9%) and cleavage rate (36.7% vs. 49.9%) for oocytes derived from 7.5 months to term bovine fetuses than for those from adult cows, respectively.

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These results were attributed to the higher proportion of fetal oocytes (12%) which remained at the GV and metaphase I (M-I) stages than was observed for oocytes from adult cows after 24 h of in vitro maturation (2.3%). These studies show that the age of oocyte donor is a significant factor affecting the developmental competence of the oocyte. Differences between oocytes from calves and cows have been found with regard to size, ultrastructure, metabolism and cytoplasmic maturation (DUBY et al.

1996; DE PAZ et al. 2001; STEEVES and GARDNER 1999; SALAMONE et al.

2001).

Oocytes from prepubertal cattle were significantly smaller (113.3-119.7 µm) than those obtained from adult cows (117-125 µm), representing a difference of approximately 5% in diameter and 10% volume (DUBY et al. 1996; GANDOLFI et al.

1998; STEEVES and GARDNER 1999). Despite this fact, maturation rates after 24 hr of culture of oocytes from 4 and 7 months old calves (75-76%) and cows (78-86%) were similar (DAMIANI et al. 1996; STEEVES and GARDNER 1999). However, it has been clearly demonstrated that oocytes with a diameter >120 µm have a high maturation rate and developmental competence to form blastocysts (LONERGAN et al. 1994; FAIR et al. 1995).

Studies employing transmission electron microscopy reveled that oocytes postmaturation from calves showed a delay in migration and redistribution of the organelles as mitochondria, lipid vesicle and cortical granules, which remain associated in large aggregates instead of dispersing evenly below the plasma membrane, suggesting a compromised cytoplasmic maturation (DAMIANI et al.

1996). A lower mitochondrial population in calf oocytes than those from cows, suggests a lower energy metabolism in calf oocytes (DE PAZ et al. 2001).

GANDOLFI et al. (1998) observed that oocytes from 10- to 14-weeks old calves metabolized pyruvate and glutamine during the first 3 hr of in vitro maturation (IVM) at lower rates than adult oocytes. Similarly, STEEVES and GARDNER (1999) and GANDOLFI et al. (1998) reported a lower oxidative metabolism of pyruvate and glutamine and a delayed uptake of glucose in oocytes from 3-4 months old calves. In another report, STEEVES et al. (1999) observed a lower glucose uptake (1.5 pmoles/embryo/hr) in 2- to 4-cell stage embryos from calves 5-7 months of age than

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from their adult counterparts (3.0 pmoles/embryo/hr), but it was equivalent at 8- to 16-cell stage and blastocysts.

Metabolic studies of ovine oocytes also have demonstrated differences between oocytes derived from 6- to -8-week-old lambs and those from adult ewes (O'BRIEN et al. 1996). The metabolism of radiolabelled glutamine by prepubertal lamb oocytes was significantly lower than that of oocytes from adult ewes. The difference was observed irrespective of whether the oocytes were matured in vitro or in vivo. No differences were observed in other metabolic parameters including oxidation of glucose or pyruvate.

Reprogramming of protein synthesis plays an essential role in control of meiotic division, and in the preparation of the mammalian oocyte for fertilization. Synthesis of proteins appears to be compromised in calf oocytes. GANDOLFI et al. (1998) observed a significant decrease in protein synthesis, measured by [³⁵S] methionine and [³⁵S] cysteine incorporation after 9 hr of IVM in calf oocytes. Differences were also found in protein patterns. Thus, proteins of 405, 146, 101 and 77 kDa were more abundant in cow oocytes after of the first 3 hr of IVM than in calf oocytes. In a similar study, KHATIR et al. (1998a) determined significant differences in protein profiles of constitutive and de novo synthesized proteins during the first 20 hr of IVM, suggesting that protein synthesis could be a reason of the lower developmental competence of calf oocytes. Furthermore, LEVESQUE and SIRARD (1994) observed similar patterns of constitutive proteins in “defective” oocytes (oocytes with dark or expanded cumulus or not fully surrounded by cumulus cells) derived from cows and in oocytes derived from less than 40 day old calves. In this study, the absence of several constitutive proteins was also observed in cumulus cells from calf follicles.

These proteins may be important for the initiation of the cascade of events necessary for calf oocytes to develop normally. The existence of an oocyte–granulosa cell regulatory loop essential for normal follicular differentiation resulting production of an oocyte competent for fertilization and embryogenesis, has been proposed (EPPIG 2001). On the other hand, it has been reported that immature calf oocytes have a nucleolus that contains two electron-dense ovoid structures encapsulated by less electron dense fibrils, whereas immature cow oocytes possess only one structure

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(DAMIANI et al. 1996). The latter is characteristic of oocytes that have completed their RNA synthesis (FAIR et al. 1996). The presence of two fibrillar structures in calf oocytes may indicate incomplete nucleolar maturation (DAMIANI et al. 1996).

Biochemical changes and physiological events have been used as parameters to study cytoplasmic maturation in calf oocytes. Some examples are the activity of MPF and MAPK (involved in the resumption of meiosis) and the relative amount of inositol 1, 4, 5-triphosphate receptor (IP3R) which is involved in the generation of calcium oscillations during fertilization. A low activity of MPF and MAPK which can be measured by the amount of phosphorylation of histone H-1 kinase was observed.

The relative amount of IP3R as determined by Western blotting was lower in oocytes derived from 6 months old calves than in oocytes derived from adult cows (SALAMONE et al. 2001).

The rate of cleavage and the rate of blastocyst formation have been used to evaluate cytoplasmic maturation after nuclear transfer, after parthenogenetic activation and after stimulation by culture in follicular fluid. A lower blastocyst rate was obtained when nuclei derived from 30-50 cell morula stage embryos produced in vitro from adult oocytes were transferred to recipient ooplasm from prepubertal calf than when nuclei from the same embryos were transferred to ooplasm of oocytes collected from adult cows (MERMILLOD et al. 1998). Similarly, embryos reconstructed by fusion of M-II chromosomes from the oocytes of adult cows into calf ooplasm cleaved at a lower rate and produced fewer blastocysts than did those reconstructed by the transfer of M-II chromosomes from immature calves into adult ooplasm (SALAMONE et al. 2001). Similar results have been obtained with oocytes from prepubertal gilts (IKEDA and TAKAHASHI 2003). Both, cleavage and blastocyst rates were significantly lower in parthenogenetically activated calf oocytes than in activated adult cow oocytes (SALAMONE et al. 2001). Calf oocytes produced fewer blastocysts than cow oocytes in culture supplemented with cow follicular fluid or fetal calf serum, indicating that prepubertal oocytes are unable to respond or do not possess the receptors for specific factors present in serum and follicular fluid that probably stimulate the developmental competence in adult oocytes (KHATIR et al. 1997).

These findings suggest that cytoplasmic maturation is compromised in calf oocytes

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affecting the synthesis and storage of upstream regulators or key regulatory components for embryonic development.

No differences between calf and cow blastocysts have been observed with respect to constitutive protein synthesis, de novo protein synthesis or total cell number (KHATIR et al. 1998b; STEEVES et al. 1999). However, differences were observed in the viability of these embryos after transfer. Pregnancy rates of 0 to 22% have been reported after the transfer of blastocysts derived from calf oocytes as compared to 39% pregnancy after the transfer of blastocysts produced from adult cow oocytes (KHATIR et al. 1998b; RICK 1996). On the other hand, ARMSTRONG et al. (1997) reported an ultrasound confirmed 45-day pregnancy rate of 43% in 23 recipients of fresh IVF embryos from calf oocytes. These pregnancies eventually resulted in the birth of seven calves (33% of embryos transferred).

REVEL et al. (1995) reported that rates of initial pregnancies determined by progesterone level 21 days of the estrous cycle after transfer of IVF embryos produced from calf oocytes were similar to the rates obtained with adult cow oocytes, (66% and 65% respectively). However, only one of the nine recipients (11%) of embryos from the FSH treated calves maintained pregnancy and delivered a full-term calf indicating high embryo losses for blastocysts derived from prepubertal calves.

The low uptake of energy substrates, low activity of MPF and MAPK as well as low rate of protein synthesis indicate that cytoplasmic maturation with its essential biochemical changes is compromised in calf oocytes. This does not prevent completion of meiosis but does diminish the ability of these oocytes to produce blastocysts at rates observed for adult cow oocytes.

Age also affects the composition of follicular fluid. DRIANCOURT et al. (2001) determined a reduced level of estradiol, low aromatase activity and differences in protein profiles in follicular fluid of healthy follicles from 3 months old calves in comparison to those from cows. However, increased blastocysts yield of cow oocytes were found in cultures supplemented with calf follicular fluid (KHATIR et al. 1997).

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