Prepuberal calves as oocyte donors: Promises and problems

ELSEVIER

Prepuberal Calves as Oocyte Donors: Promises and Problems R.T. Duby’,

P. Damiani’, C.R. Looney’, R.A. F&ore’

and J.M. Robl’

’ Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01002 USA ‘TrarrsOva Genetics, Sioux Center, IA 5 1250 USA ABSTRACT The use of prepuberal heifers as oocyte donors in breeding programs could decrease the generation interval in cattle and increase the genetic rate of gain. While large numbers of follicles develop in response to exogenous gonadotrophins the oocytes they contain lack developmental competence until the animals are 6 to 8 months old. In vitro fertilization rates are normal but rates of cleavage and subsequent development are low. The oocytes are smaller than those collected from adult cattle and the cortical granules fail to disperse evenly during maturation. Oocytes from prepuberal calves release less calcium in response to challenges with InsP3. The lower peak height and altered pattern of Ca*++ oscillations when compared to oocytes of mature cows suggests that cytoplasmic maturation is incomplete in heifers 175 days of age or less. However, the development of ultrasound guided methods for the recovery of oocytes from calves greater than five months old and their improved developmental capacity as demonstrated by development to blastocysts and birth of live calves indicates that the prepuberal calf will play a significant role in animal breeding programs of the future by serving as sire dams or by expanding the influence of genetically superior animals in producer herds. Keywords: prepuberal calf, oocyte, maturation, calcium release INTRODUCTION Genetic gain in production traits of dairy and beef cattle is being limited by the length of the generation interval (GI). The effect of the GI on genetic gain is readily apparent from the following equation:

Intensity of Selection x Accuracy of Selection x Genetic Variance Annual Genetic Gain = ________________________________________________________________________-__ Generation Interval Gains in genetic merit during the last 20 years are due predominantly to the increases in intensity and accuracy of sire selection made possible through the use of artificial insemination and progeny testing programs. Increases in intensity of selection of sire dams was made possible through embryo transfer (ET) and more recently by retrieval of oocytes by ultrasound guided follicular aspiration and their subsequent in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC; IVMFC) and transfer. However, to be effective in identifying animals of truly superior genetic merit at least 50 viable oocytes would have to be recovered from each of 12 daughters and fertilized with semen from at least 12 bulls in a factorial mating system (70). The time required to complete such a trial would only minimally change the generation interval. As an alternative using oocytes from fetal gonads has been Acknowledgements Supported in part by U.S.D.A. grant (RAF) and Mass. Ag. Exp. Sta. Project 620 (NE-161). expertise of J. Balise in collecting oocytes and C. Long in evaluating oocytes is appreciated.

Theriogenology 45: 121-l 30, 1996 0 1996 by Elsevier Science Inc. 655

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0093-691X/96/$15.00 0093-691X(95)00361-4

The

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Theriogenology

suggested (9) and significant benefits have been identified if oocytes from prepuberal heifers could be used with marker assisted selection (28). Combining a MOET scheme with IVF of calf oocytes was predicted to increase annual genetic gain 22% over conventional progeny testing programs (41). The purpose of this review is to discuss follicular development, oocyte maturation, fertilization and early embryonic development in prepuberal heifers and assess their possible use in cattle breeding programs. Follicular development and turnover in prepuberal heifers Vesicular follicles appear in the fetal bovine ovary between 220 and 240 days of gestation. rNeonata1 ovaries contain from 75,000 to 300,000 oocytes and follicles up to 5mm are present as early as day 5 after birth (23). Ovarian weights increased from 0.5g at two weeks of age to 4g at 4 months and log by 12 months. The number of follicles also increased and by 7 weeks a constant pool of approximately 50 vesicular follicles was present. Approximately 50% of these were atretic (8)and only 39% of oocytes collected from 4 month old calves were viable (67). Numerous studies (3,4,29,53,58) including our work (15,16,17,43) shows that these follicles can be stimulated to grow with exogenous gonadotrophins. While IVF rates (sperm penetration) are similar to those of oocytes recovered from adult cattle, cleavage and development rates are low suggesting that maturation of the ooplasm and/or nucleus is incomplete. Lack of developmental competence in oocytes of prep&era1 animals is not restricted to the bovine as it has been reported in the mouse (21), However, normal fertilization rates and development to goat (37,50) and sheep (11). blastocysts of oocytes collected from prep&era1 calves (4) and sheep (5,19) has been reported. The reasons for these differences are not known. Follicular development in mature cycling cows and heifers occurs in 2 to 4 waves during the estrous cycle (54,60). Dominant follicles developing in the initial waves become atretic while one follicle of the last wave becomes dominant and ovulates following regression of the corpus luteum (CL). The superovulatory response, the number of ovulations and viable oocytes/embryos produced, is related to when treatment is started during the wave (3,lO). The variability of the superovulatory response and the morphology of the ovaries of the prepuberal heifer (3) suggests that follicular waves develop prior to puberty. This is further supported by the observation that plasma estradiol-17beta (E2) levels in gonadotrophin stimulated calves varied and were correlated to the number of follicles present at the time of the initial injection of FSH (65). More recently, follicular waves were identified in heifers beginning at 2 weeks of age (24) and waves observed at 8 months of age continued to first ovulation at 12 months (1). While the follicles were qualitatively similar to those of adult cattle they failed to ovulate, were smaller and had shorter growing and static phases that resulted in shorter interwave intervals. Wave emergence was associated with an increase in FSH and LH similar to those occurring in mature heifers (2). However, all follicular cycles, natural as well as those induced by exogenous gonadotrophii are anovulatory unless exogenous I.&I is administered. A possible explanation of this phenomenon is that levels of circulating E2 are not high enough to elicit the LH surge required for ovulation. That ovulations can be induced by administration of LH or hCG (58) and corpora lutea (CL) form downstream from the and secrete progesterone (61) suggests that mechanisms pituitary-hypothalamic axis are functio~l. Further support for this hypothesis the response of heifers 3, 6, and 9 months of age to G&I-I (7.51). Heifers in all age groups released FSH and LH in a manner similar to adult cattle. Basal levels of FSH and LH were also similar but LH pulses did not occur until the 9th month. Intraovarian substances may inhibit the pulsatile release of LH. Unilateral ovariectomy as early as three weeks of age resulted in a temporary increase in the frequency and amplitude of FSH and LH release (38) and that they could be blocked with charcoal stripped follicular fluid lends further support to the hypothesis.

Theriogenology

Functional

characterization

123 of follicles and oocytes from prepuberal

heifers

Follicle size in gonadotrophin stimulated prepuberal calves is smaller (lo-12mm) than those in adult cows (H-20). Preliminary observations indicate that development of the granulosa layer is normal but expansion in response to exogenous hCG may be delayed (Tables 1,2). An increase in progesterone (P) and decrease in E2 occurred as the cumulus expanded (Table 1). Germinal vesicle oocytes were recovered from “estrogen active follicles” while metaphase II oocytes were recovered from follicles producing predominantly P (Table 1). While the ooplasm of the majority of the oocytes was homogeneous, they were significantly smaller ( 119.7umk1.43) than those obtained from cows (1256um f3. While this represents a difference of approximately 5% in diameter it represents a difference of 10% in volume. It would appear that there is an uncoupling of the expansion of the granulosa cells and the growth and maturation of the oocyte within. Oocytes smaller than 120uM fail to complete maturation (56), but increasing the length of maturation increased the number of oocytes maturing (52). It is possible that protein and mRNA synthesis are incomplete in the smaller oocytes. While some are capable of completing meiosis, fertilize and cleave, they lack the proteins and/or mRNA required for developmental competence. This argument is supported by the observation that constitutive expression of proteins by cow and calf oocytes are different and correlated to the developmental capacity of the oocyte (40). Protein synthesis by the cumulus cells from intact cumuli differed from those secreted by “defective” cumuli. The secretory pattern of the calf oocyte resembled that of “defective” oocytes. Protein synthesis is essential for completion of meiosis in bovine oocytes and is correlated to the synthesis of M-phase promoting factor (MPF;64). Collectively these observations suggest that prepuberal oocytes differ in their ability to synthesize proteins required for maturation and subsequent development. Table 1. Characteristics of follicles collected germinal vesicle or metaphase II oocytes. Category No follicles Mean diameter (mm) Mean volume (ml) Mean cumulus scored Mean cytoplasmic scoree Mean fol. fluid estradiol (ng/ml’) Mean fol. fluid progesterone (rig/ml-‘) Mean E/P ratio a b c d e

from prepuberal

calves containing

Germinal Vesicle : 8 0:28 1.11 1.44 950.0 24.10 1.44

+ + + f * + +

83a :04 .lO .15 15.10 8.03 .52

either

Metaphase II 8.2 0.29 2.87 1.00 14.79 97.37 0.15

Means + SEM Difference between GV and MI1 different, p < 0.08 Difference between GV and MI1 different, p < 0.05 Cumulus score: 1 =compact, 2 = partial expansion, 3 =expanded, Cytoplasmic score: 1= homogenous, 2 = granular, 3 =degenerate

y6.20 + 0.03 kO.13 kO.00 .-&6.33b + 14.60b + 0.06’

4 = degenerate

The capacity of the oocyte to become developmentally competent is related to the age of the donor. In mice, nuclear mechanisms required to direct the function of the cells of the stratum granulosum and cumulus matures between days 15 and 20 after birth (22). Similarly, development of the adenyl cyclase second messenger system in the granulosa cells of developing follicles in the prepuberal heifer was age dependent (68). Immature pig oocytes possess a meiosis inhibiting factor that is not species specific as immature pig oocytes fused to

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Theriogenology

mature mouse oocytes inhibited germinal vesicle breakdown (52). It is clear that oocytes collected from prepuberal animals differ from those of adult animals. It is implied from these observations that methods will have to be developed to promote growth of oocytes and allow them to complete the synthesis of mRNA and proteins required for maturation, fertilization and developmental competence if the young calf is to be used as a donor of oocytes for breeding programs. Plasma concentrations of estradiol-17B are elevated in the superovulated prepuberal heifer and progesterone levels are basal unless the animal is treated with hCG or LH (61) suggesting that the thecal and granulosa cells are functional. It would appear that the functional response of the granulosa/cumulus cells to LH is critical to oocyte maturation and acquisition of developmental competence as stimulation of prepuberal calves with hGG facilitated maturation in vitro and development to blastocysts (55). We have observed that development to MI1 was associated with cumulus expansion and a decrease in the ratio of E:P (Table 1). Functional changes in the cumulus and granulosa cells have been shown to important regulators of maturation in bovine oocytes (4059). Fertilization,

cleavage and developmental competence of oocytes from calves

Problems associated with in vivo fertilization and embryo collection from prepuberal heifers makes it essential that IVMFC systems be developed. Initial studies indicated that significant numbers of oocytes from prepuberal heifers matured in vitro for 22-24 hours failed to mature. The effect of in vivo maturation time on oocyte maturation (Table 2). Based on the expansion of the cumulus, increasing the time between injection of hCG and oocyte collection Table 2. Effect of interval between hCG injection and oocyte recovery on maturation in viva.’ Hours fro4mhCG to oo;{te recovery Total

28 No. calves No. follicles’ No. oocytes recovered No. mature oocytes

39212

60+ ;17

41:

20+8

42+

18+5

3

15

+2

26+

10

17 45_t7 25+-5

14

6+ 21Ot

4 (% recovered)

(14%)

(53.%)

i All calves received 24 mg FSH-P over over three days. after injection of 2000 i.u. hCG i.v. * Data presented as means + SEM

(38%)

(33 %)

Oocytes collected at specific ties

increased the number of oocytes with expanded cumuli. However, significant numbers of cumuli remained intact whiie a similar proportion were denuded when collected. These observations suggested that the population of follicles being stimulated by exogenous gonadotrophins and aspirated were in various stages of development. This observation lead US to conclude that a method to “synchronize” follicular development prior to gonadotrophrc stimulation was required. We reasoned that stimulation of follicular development with a low dose of eCG (250 i.u.) would sensitize follicles to subsequent injections of FSH-P by inducing FSH receptors. The effects of estrogens produced by the developing follicles on LH release might also be blocked by progestogens. Accordingly, beginning on day 0 of treatment heifers receive 250 i.u. eCG followed seven days later by a second injection and an implant of

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125

Norgestomet. Seven days following Norgestomet implantation, 24 mg of FSH-P was administered over 3 days (5,5,4,4,3,3mg). The number of expanded cumuli decreased from 33 % to 0.7 % and the percent denuded oocytes increased from 13.9% to 27 % and intact oocytes from 52% to 68%. These observations suggest that oocyte development may be synchronized by blocking the effects of endogenous release of LH but the population of follicles that is being stimulated (rescued) by exogenous gonadotrophins may contain oocytes that are in various stages of atresia as significant numbers of denuded oocytes were recovered. In vitro fertilization (as measured by sperm penetration) of prepuberal calf oocytes is comparable to those of adult cattle (Table 3). While the incidence of polyspermy is similar the number of “abnormally” fertilized oocytes is higher. However, the peripheral distribution of mitochondria and lipid vehicles, horizontally stacked microvilli, absence of a perivitellin space, cumulus cell processes that traversed the zona pellucida and embedded their distal tips in the ooplasm and clusters of cortical granules that were still associated with the Golgi were similar to the fine structure of immature adult oocytes (18,33-35). After maturation the cortical granules migrated to more peripheral locations, the cumulus cell processes retracted and microvilli extended into the newly formed perivitellin space. However, the cortical granules failed to disperse evenly over the cortex despite the fact that they migrated. This observation may help explain the polyspermy noted in these oocytes. Table 3. Fertilization in vitro.

of oocytes collected from prepuberal heifers and matured and fertilized Treat A N %

No. Oocytes Metaphase II 2 Pronulcei Polyspermic Abnormal

242 8 122 69 43

1.3 50.4 28.5 17.8

Treat B N % 47 6 10 ::

12.8 21.3 38.3 27.7

Treat C N %

Adult N %

95

-

431

-

51 12 30

55.8 12.6 31.6

223 125 46

51.7 29.0 10.6

‘Treat A: eCG, 250i.u. 1 week before and at time of Norgestomet implant, FSH-P 24 mg over 3 days beginning of days 7 days after second eCG. Treat B: As A and 2000 i.u. hCG administered i.v. 12 hours after last FSH-P. Treat C: 24 mg FSH-P over 3 days, 2000 i.v. hCG i.v. 12 hours after last FSH-P All ooytes collected 22-24 hours after last FSH-P or hCG. Adult: Ooytes collected from ovaries of slaughtered cows and IVM/IVF. The developmental competence of prepuberal oocytes also appears to be compromised. Approximately 22.5 % of 502 prep&era1 oocytes aspirated from follicles of calves aged 75-153 days cleaved after IVMF and only 8 (1.6%) developed to blastocysts. Lack of developmental competence may be related to the age of the calf at the time of gonadotrophin stimulation. None of the oocytes collected from calves less than 240 days of age developed to blastocysts while 60% of cleaved oocytes did so when the calves were older than 240 days and 3 of 9 blastocysts transferred resulted in live calves (43). Developmental competence was enhanced when the donors were treated with hCG prior to oocyte collection in calves 5 months or older (55). It is not clear if the differences in developmental competence between oocytes of prepuberal calves and peripuberal heifers is due to differences in the cytoplasm or nucleus. Our transmission electron microscopy studies (15,16) of oocytes removed from IVF after 8 hours of coincubation with sperm to minimize the incidence of polyspermy (42) show that sperm head decondensation, aster formation, development of the female pronucleus and

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Theriogenology

migration and apposition of the pronuclei appears to be normal. Functional studies in the mouse and hamster oocyte show that there is a redistribution of the endoplasmic reticulum (ER) during meiotic maturation (4658). The migration of the organelle to the cortical region of the cytoplasm, increasing numbers of vesicles, increased storage of Ca2++ and numbers of Inositol 1,4,5trisphosphate receptors are indicators of oocyte maturation and are required for the induction of calcium oscillations required to activate the oocyte at fertilization.

Intracellular calcium release from prepuberal calf oocytes Calcium has been recognized as an important second messenger in the fertilization We have described calcium release in IVM/IVF bovine oocytes from mature process. cows/heifers (25) and it is clear that episodic releases of Ca 22++ are induced by the fertilizing sperm. Increases in cytosolic calcium have been implicated in cell cycle regulation (36,44,70) and the release of cortical granules to induce the block to polyspermy (63). Increases in cytosolic calcium are mediated by activation of G-proteins which cause hydrolysis of membrane bound phosphoinositol (4,5)-bisphosphate (PIP2) by phosphoinositidase C with the production of diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (InsP3). DAG activates phosphokinase C (PKC) while InsP3 binds to receptors on the endoplasmic reticulum (ER) and induces release of calcium (InsP3 induced calcium release (IICR). Calcium can also be released from InsP3 insensitive stores by elevated levels of cytosolic calcium (calcium induced calcium release, CICR). Release from these stores can be stimulated by ryanodine agonists. Intracellular release of calcium during fertilization of hamster oocytes is due to InsP3 (47-49) and inhibition of PIP2 hydrolysis prevented resumption of meiosis in bovine oocytes (30). It is clear that calcium plays a pivotal role in regulating the events associated with oocyte maturation by activating cellular processes required for normal development. Table 4. Induction of intracellular calcium release by microinjection collected from prepuberal heifers and adult cows.

of InsP3 into oocytes

5.0 uM 11rsP3~ No. oocytes

cow calf

7 8

Baseline intracellular free Ca+ +

cow calf

131.0 + 30.8b 131.6 + 27.5

Increase in intracellular concentration of free Ca+ +

cow calf diff

530.6 + 91.9 69.6 + 52.1 461.v

Duration of rise (set)

cow calf diff

97.2 + 14.1 48.8 + 7.8 48.4”

L Concentration of InsP3 in microinjection pipet. Final concentration 1% of initial concentration in pipet. b Intracellular free calcium, Mean + SEM, uM. c Difference between cow and calfsignificantly different, p < .OOl.

in oocyte approximately

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Development of calcium storage/release mechanisms has been shown to be dependent upon the stage of maturation in starfish oocytes (13). The high incidence of polyspetmy and poor development to blastocysts of oocytes obtained from prep&era1 calves may be due to delayed migration of cortical granules and development of calcium release/storage mechanisms. Initial experiments comparing the effect of micromjecting 500 uM InsP3 (a concentration that releases maximum amounts of intracellular Ca2++) into cow or calf oocytes showed that were no differences in the amount of calcium released. These data suggested that calcium stores were similar in the two groups of oocytes. However, when threshold amounts (5uM) were injected significantly less calcium was released from the calf oocytes (Table 4). In addition to a lower peak height, the time required for intracellular calcium levels to return to baseline values was significantly shorter in the calf oocytes. Similar age effects have also been reported in mice (27,45). It is clear that IICR calcium release in the prepuberal calve oocyte is different from the adult oocyte. In all species, a series of calcium oscillations are required to initiate intracellular events associated with cleavage and subsequent development of the embryo (25,39,48,62). Preliminary studies also indicate that Ca*+ release by sperm fertilizing prepuberal oocytes is lower than in oocytes from adult cattle. When bovine oocytes are arrested at metaphase II are electrically stimulated in a calcium containing solution, intracellular Ca*+ + increases and induces parthenogenic development (14). Histone H-l kinase activity was suppressed and correlated to a decrease in MPF. A single pulse resulted in a transient decreases in MPF while multiple pulses suppressed MPF long enough for activation to be completed. The sperm induced series of calcium spikes in bovine oocytes (25) are initiated primarily by InsP3 (26) as release could be blocked by heparin. It has also been suggested that the height of the fist Ca*” peak may determine if periodic release will follow (71). Yue et al., (71) demonstrated that caffeine (agonist of the ryanodine receptor) ryanodine or cyclic ADP-ribose (intracellular modulator of CICR) all stimulated release of calcium in IVM bovine oocytes. The effects of caffeine and ryanodine could not be blocked by heparin suggesting that the InsP3 receptor was not involved. In human oocytes it appears that increases in intracellular calcium initially occur in the cortex of the oocyte followed by an explosive release from the center (66) suggesting that both IICR and CICR mechanism are required for oscillationsRegardless of the mechanism involved in the release of Ca*++ our data indicate that it is immature in prepuberal heifers. Since we showed that Ca*” stores were similar in prepuberal and mature cattle oocytes, it would appear that fewer InsP3 receptors are present on the surface of the storage vesicles in calf oocytes or they are less sensitive to stimulation by InsP3. Collectively, these data suggest that while follicular development can be induced by exogenous gonadotrophins in prepuberal calves that oocyte development is delayed. The smaller size of the oocytes, different pattern of protein synthesis and altered pattern of calcium release make it clear that new methods will have to be developed to stimulate oocyte growth and maturation. hture

prospects

The very young calf as an oocyte donor will await development of new methods of maturation. Identification of follicular waves two weeks after birth and oocyte collection by laparoscopy (4) and ultrasound guided follicular aspiration (12) offers the potential of a heifer having several daughters calving at about the same time that she does for the first time. Until we better understand oocyte maturation and develop new methods for IVM the use of calves that are 6 months of age and older is a more practical alternative. Follicular waves persist in these animals, developmental competence can be induced in vivo with hCG and oocytes can be collected by ultrasound guided follicular aspiration. The incorporation of these technologies into MOET schemes to produce/identify future sire dams and expand desirable genotypes in producer herds offers a viable method to decrease the GI and increase genetic gain.

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