Amino acid transport in mammalian oocytes

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0014.4827/79/040333-09$02.00/O

Experimental

AMINO

ACID

Cell Research 119 (1979) 333-341

TRANSPORT

IN MAMMALIAN

OOCYTES

R. M. MOOR’ and M. W. SMITH2 Agricultural Cambridge

Research Council Institute of Animal Physiology, ‘Animal Research Station, CB3 OJQ and ‘A.R.C., babraham Hall, Babraham, Cambridge CB2 4AT, UK

SUMMARY Uptake of “C-labelled amino acids into single oocytes was determined using 3H-labelled choline to correct for extracellular space. Cycloleucine, a non-metabolisable amino acid sharing an entry mechanism with methionine and phenylalanine, was transported in accord with Michaelis-Menten kinetics. At extracellular levels below 8 mM, cycloleucine was concentrated within the oocyte. The proportion of sheep oocytes having a functional amino acid transport system (i.e. cycloleucine flux > 1 nmole crnm2h-l) was highest in pre-ovulatory follicles (97%), and lowest in atretic follicles (59%). Amino acid fluxes in functional germinal vesicle oocytes were similar at all stages of development studied. An increase in V,,, but not K, during meiotic maturation resulted in a doubling of amino acid uptake in metaphase II oocytes. These increased fluxes were under gonadotropic regulation and were independent of nuclear maturation. Amino acid uptake by mouse oocytes was approximately half that measured in sheep oocytes.

Mammalian oocytes reach prophase of the first meiotic division and then enter a period of prolonged meiotic arrest. Immediately before ovulation some oocytes resume meiosis and undergo a series of structural and synthetic changes resulting in the full maturation of the oocyte. These changes include a relocation of intracellular organelles [l], a cessation of RNA synthesis [2] and specific qualitative and quantitative changes in polypeptide synthesis [3, 41. Changes in the oocyte membrane parallel these intracellular events. Gap-junctional complexes between the oolemma and surrounding follicle cells become disrupted [5, 61, electrophysiological and ionic properties of the membrane change [7,8] and amino acid uptake into the mouse oocyte increases [9]. It has been our object to extend these observations by describing the characteristics of amino acid entry into mammalian 22-791805

oocytes examined at different stages of development and maturation. The majority of experiments have been carried out on sheep oocytes but for comparative purposes flux rates in mouse oocytes have also been determined.

METHODS Tissue preparation Ovaries were obtained from sheep (i) during the luteal phase of the oestrous cycle; (ii) 40 h after the subcutaneous injection of 1200 IU pregnant mare serum gonadotropin (PMSG); or (iii) 18 h after the preovulatory release of luteinizing hormone (LH) induced by the sequential administration of PMSG, prostaglandin and gonadotropin-releasing hormone [lo]. Intact follicles were dissected from the ovaries and classified into uniform groups according to folhcular size and degree of atresia [ 111. Mouse oocytes were obtained by injecting unmated CFLP females with PMSG and human chorionic gonadotropin (HCG) according to the method of Donahue [12]. Oocytes were recovered by dissection 10 h after administration of HCG. Exp Cell Res 119 (19791

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Moor and Smith

Table 1. Effect of follicle

cells, stage of oocyte development and type of radioactive label on the measurement of extracellular space in single oocytes after 2 h incubation in vitro Mean (+S.E.M.) extracellular space (~1 cm-*) Choline chloride

Inulin

Follicle cells

Stage of oocyte development

No. oocytes

Space marked

No. oocytes

Space marked

Absent

Germinal vesicle Metaphase II Germinal vesicle Metaphase II

128 65 25 57

2.23f 0.15 2.07+ 0.17 57.76+ 12.66 19.43* 4.84

8 38 19 36

lXiOto.% 1.53kO.28 0.92+0.18 2.47kO.60

and incubation

of oocytes

Present

Preparation

Follicles were opened, washed free of follicular fluid and the oocyte and surrounding cumulus cells dissected out. The subsequent handling of oocytes was carried out at 37°C in a medium consisting of Dulbecco’s phosphate-buffered saline supplemented with bovine serum albumin (4 mg ml-‘) and energy source (pyruvate 0.36 mM; lactate 23.8 mM, and glucose 5.5 mM). Cumulus cells were removed mechanically from sheep oocytes using a number of finely graded micropipettes. Mouse oocytes were denuded of cumulus elements using hyaluronidase (150 IU ml-’ for 6 min) followed by extensive washing in medium to remove the enzyme. After completion of the preparative procedures, oocytes were placed in wells of microtitre test plates and incubated for varying intervals in a medium containing a 3H-labelled marker of extracellular space together with 14C-labelled amino acid. The incubation medium consisted of Dulbecco’s phosphate-buffered saline, together with the energy source described above; bovine serum albumin was not included, since preliminary tests had demonstrated that it was without effect on amino acid uptake. Incubations were terminated by transferring single oocytes from the labelled medium into separate Petri dishes containing 4 ml Dulbecco’s phosphate-buffered saline at 4°C. Each oocyte was washed for 6-10 set in this medium before being placed on a coverslip, all excess medium removed, and 10 ~1 lysis buffer added [ 131.Conventional counting techniques were used and appropriate corrections made for dual-channel counting. The average spillover of 14Ccounts into the aH channel was 15%. There was no spillover of 3H counts into the ‘*C channel. Quenching due to the lysis buffer was less than 2 %.

Isotopes The following isotopes were purchased from the Radiochemical Centre, Amersham, Bucks: [3H]inulin, >300 mCi/mmol; [methyl-3H]choline chloride, 5-15Ci/ mmol; o-l[l-3H]gaIactose, 5-20 Cilmmol; l-aminocyclopentane-l[l~]carboxylic acid (cycloleucine), 4060 mCi/mmol and u-gIucose[l-“C]lactose, 22 mCi/ mmol. Polyethylene glycol (mol. wt 4000), 0.5-2 Exp CdRes 119f1979)

mCi/g and [I-aH(N)mannitol, >3 Ci/mmol were purchased from New England Nuclear, Boston, Mass.

RESULTS Extracellular

space markers

The zona pellucida surrounding the mammalian oocyte constitutes an extracellular space which will become equilibrated with medium containing labelled amino acid during the course of an incubation. Counting the radioactivity in these oocytes without washing will overestimate the true uptake by an amount equivalent to the extracellular space. Washing oocytes prior to counting is likely to cause the eventual removal of all counts from the zona pellucida, for this region has been shown to be freely permeable to many substances of low molecular weight [14]. There is, however, no way of telling whether such washing might remove radioactivity from the oocyte itself. Because of this it was felt desirable to wash as little as possible and to rely on an independently labelled space marker to correct for extracellular space. In preliminary studies single denuded oocytes at the germinal vesicle stage of development were incubated for 2 h with 3H- or 14C-labelled inulin, choline chloride, polyethylene glycol, mannitol, galactose or lactose. From the results of those studies, inulin and choline chloride

Amino acidfluxes in oocytes

0’

60

180

300

time (min); ordinate: extracellular space (~1 cme2). m-m, Inulin; O-O, choline chloride. Mean size (fS.E.M.) of the extracellular space in sheep oocytes denuded of follicle cells and incubated for 30-300 min using 3H-labelied inulin or choline chloride as space markers. Each point represents 13186determinations.

Fig. 1. Abscissa:

appeared most suitable for further investigation. The effect of the stage of oocyte maturation and the presence or absence of follicle cells on the size of the space marked by these two compounds is shown in table 1. In the absence of follicle cells the size of the extracellular space was not significantly affected by either the stage of oocyte development or the compound tested. By contrast, the presence of follicle cells greatly increased the apparent size of the choline space in germinal vesicle and to a lesser extent metaphase II oocytes (P
335

An acceptable space marker should mark a constant space irrespective of the length of the incubation period. That this requirement is adequately met by both choline chloride and inulin when tested on denuded oocytes is shown in fig. 1. In addition, inulin showed a constant space with time when tested with cumulus-clad oocytes; the corresponding relationship using choline chloride between extracellular space and incubation time in cumulus-clad oocytes was not determined. Since choline chloride (mol. wt 139.5) marked a slightly larger space than the bigger inulin molecule (mol. wt 3 000-5 000) it was considered more prudent to use choline as the extracellular marker for all experiments using denuded oocytes. Inulin was used for all experiments in which cumulusclad oocytes were studied. Amino acid uptake into single sheep oocytes Oocyte variability. The use of single oocytes in the present study allows one to determine the degree of variability in amino acid fluxes in an apparently homogenous population of germinal vesicle oocytes obtained from a single class of large (3.54.5 mm diameter) non-atretic follicles. The distribution of oocytes, plotted as a function of amino acid uptake after 1 or 4 h incubation in 0.4 mM cycloleucine, is shown in fig. 2. After 1 h incubation, uptake in 13 of the 133 oocytes (9.8%) was below 1 nmole cm-* while the mean uptake was 5.01f0.27 nmoles cm-* h-’ (n=133). After 4 h incubation, cycloleucine uptake in 12.7 % of oocytes was still below 1 nmole cm-*; the highest uptake in that sub-group (deficient transport group) was in fact only 0.46 nmoles cm-* whilst the overall mean uptake was 11.16+0.87 nmoles cm-* 4 h-’ (n =46). It is concluded that a proportion of E.rpCd/ResI19

(1979)

336

Moor and Smith

30 I

A

n

Fig. 2. Abscissa: cycloleucine uptake (nmoles cm-*); ordinate: no. of oocytes. Distribution of sheep oocytes (A) on the basis of

their ability to transport amino acid during a 1 h incubation in 0.4 mM cycloleucine; (B) according to their ability to transport amino acid during a 4 h incubation in 0.4 mM cycloleucine.

oocytes were deficient with respect to their capacity to transport cycloleucine. In all subsequent experiments oocytes were excluded from further consideration if their cycloleucine uptake was less than 1 nmole cm-* h-l. It was of considerable interest to examine

the relationship between the proportion of oocytes with deficient cycloleucine uptake and the class of follicle from which they were derived (table 2). Oocytes obtained from follicles in the secondary and tertiary stages of atresia had deficient cycloleucine uptake in 41% of instances whereas only 3 % of oocytes from the heavily selected non-atretic preovulatory follicle population showed deficient uptake. The 11% of oocytes with deficient uptake in the large non-atretic group of follicles and the 23 % in the small non-atretic group corresponds reasonably closely with the error associated with the macroscopic selection of nonatretic large and small follicles [I 11. It is concluded that a close correlation exists between the extent’ of atresia in the follicle population and the proportion of oocytes showing deficient uptake. Effect of follicle cells on amino acid uptake. Cross & Brinster [9] found that the presence of cumulus cells increased leucine uptake by mouse oocytes in the germinal vesicle stage, decreased it at metaphase I and were without effect on uptake when measured at the metaphase II stage of development. To examine this relationship

Table 2. The effect offollicle class on the number of sheep oocytes showing a deficient rate of uptake of 0.4 mM ‘Clabelled cycloleucine Oocytes with fluxes of cycloleucine of less than 1 nmole crnm2h-’ have been defined as showing ‘deficient cycloleucine uptake’ Oocytes showing deficient cycloleucine uptake Follicle classification Large non-atretic (3.5-5.0 mm diam.) LH activated (5.0-8.0 mm diam.) Small non-atretic (2.G3.5 mm diam.) Grossly atretic (3.5-5.0 mm diam.) PMSG stimulated (3.5-7.0 mm diam.) Exp Cell Res 119 0979)

Oocyte nuclear configuration

Oocytes (total no.)

No.

%

Germinal vesicle

180

19

11

Metaphase II

124

4

3

Germinal vesicle

56

13

23

Germinal vesicle

17

7

41

Germinal vesicle

59

10

17

Amino acidfluxes

in oocytes

337

Table 3. Effect offollicle

cells on amino acid uptake by sheep oocytes incubated in 0.4 mM cycloleucine for a period of 2 h Follicle classification

Oocyte nuclear configuration

Follicle cells

Oocytes (total no.)

Mean f S.E.M. cycloleucine uptake (nmoles cmez 2 h-‘)

Large non-atretic (3.5-5.0 mm diam.) LH activated (5.0-8.0 mm diam.) PMSG stimulated (3.5-7.0 mm diam.)

Germinal vesicle

Present Absent Present Absent Present Absent

19 37 18 29 12 18

6.87kO.53 7.85+0.48 15.14k1.79 11SOk0.66 12.04f2.95 10.83+0.66

Metaphase II Germinal vesicle

further, sheep oocytes, with and without follicle cells, were incubated for 120 min in the presence of 0.4 mM cycloleucine with 3H-labelled inulin as space marker. The results, presented in table 3, show (i) that follicle cells did not significantly affect cycloleucine uptake in germinal vesicle oocytes; (ii) that they had a marginally stimulating effect on uptake in metaphase II oocytes (t=2.165;df=45;P=0.05) and that they had no effect on PMSG-treated oocytes. The variability in uptake within groups of oocytes enclosed by follicle cells was always greater than in corresponding groups of denuded oocytes. This variability was due to exceptionally high rates of uptake in a small number of oocytes enclosed by follicle cells. The conclusion drawn from the present

results is that follicle cells have at most a marginal effect on amino. acid uptake and then only in metaphase oocytes. Oocytes enclosed by follicle cells were not used further in these studies. Uptake of natural and artificial amino acids. There are considerable advantages in

using a non-metabolizable amino acid in uptake studies provided always that the artificial amino acid is transported on the same system as the natural compound. Comparative and competitive studies, summarized in table 4, were carried out to determine the extent to which cycloleucine fluxes resembled those of methionine and phenylalanine. The uptake of cycloleucine by germinal vesicle oocytes was approximately three times that for either of the natural amino acids. Methionine uptake did not dif-

Table 4. Comparative and competitive studies on the rate of uptake and percentage inhibition induced by incubating sheep oocytes at the germinal vesicle stage with different amino acids % inhibition (5 mM amino acid)

Substrate amino acid (0.4 mM)

Uptake (nmoles cm-* h-l)

Methionine

1.40*0.13 (24) 1.65kO.23 (14) 4.44f0.36 (13)

Phenylalanine Cycloleucine

Methionine

Phenylalanine

-

-

-

40

&O.ool)

(PCO.02) Exp Cell Res I1 9 (1979)

338

Moor und Smith

Fig. 3. Abscissa: time (mm); ordinate: cycloleucine uptake (nmoles cmWz). O-O, Untreated germinal vesicle oocytes; Cm, metaphase II oocytes; A-A, PMSG-treated germinal vesicle oocytes. Mean uptake (fS.E.M.) of amino acid by (A) sheep oocytes obtained at the germinal vesicle stage (0) and 18 h after the resumption of meiosis (B; metaphase II oocytes); (B) by metaphase II oocytes (W) and germinal vesicle oocytes (A) obtained from sheep treated with gonadotropin (PMSG) 36 h previously. Oocytes in both experiments were incubated in 0.4 mM i4Clabelled cycloleucine with 3H-labelled choline chloride as space marker. Each mean value represents 8-49 separate determinations.

fer significantly from that of phenylalanine. The presence of 5 mM methionine or phenylalanine reduced the uptake of cycloleucine by 74 and 40% respectively (PC 0.001 and <0.02), thereby establishing that the three amino acids share a common entry mechanism. This, together with its faster rate of uptake and resistance to metabolic degradation, made cycloleucine the amino acid of choice for subsequent studies. Amino acid fluxes in sheep oocytes at various stages of development vesicle oocytes. Oocytes in meiotic arrest, obtained from three entirely different classes of antral follicles, were incubated for varying lengths of time in 0.4 mM cycloleucine. No difference was observed in uptake rates of cycloleucine in oocytes from the three classes of follicles, namely small immature, fully developed and grossly atretic follicles. It must, however, be borne in mind that the proportion Germinal

Exp Cell Res 119 (1979)

15

5

l

L 0

5

10

I5

Fig. 4. Abscissa:

cycloleucine corm. (mM); ordina?e: (A) uptake (nmoles crnm2h-r); (B) R, ratio of cycloleucine concentration in oocyte and medium. (A) Concentration dependency of amino acid uptake by germinal vesicle (A) or metaphase II (0) sheep oocytes incubated for 1 h in media containing different concentrations of cycloleucine. Values are means (*S.E.M.) of from 4 to 31 separate determinations. The hyperbolic lines are calculated using the iterative procedure of Bliss & James [IS]. (B) Ability of metaphase II oocytes to concentrate cycloleucine plotted against cycloleucine concentration in the culture medium. The interrupted line represents the line of equilibration below which no concentration takes place.

of oocytes with deficient amino acid transport, excluded from these calculations, is directly related to the type of follicle from which the oocytes were derived (see table 2). It can, therefore, be concluded that although the class of follicle determines the number of germinal vesicle oocytes with a functional transport system it does not influence flux rates into functional oocytes. Metaphase ZZ oocytes. Gonadotrophins secreted before ovulation initiate a wide range of nuclear and cytoplasmic changes in the oocyte. Possible alterations in amino acid transport during maturation have been measured by comparing cycloleucine up-

Amino acidjluxes

take into germinal vesicle oocytes with that obtained using oocytes 18 h after the induction of gonadotrophin release (metaphase II oocytes). The results illustrated in fig. 3a show that the rate of uptake doubles after gonadotrophin treatment. This increase in uptake is not, however, dependent upon resumption of meiosis. It can also be induced by injection of PMSG into sheep 36 h prior to collection of ovaries (fig. 36). The rate of cycloleucine flux into germinal vesicle oocytes taken from PMSG-treated sheep are significantly higher (PcO.01) than those from untreated animals and are comparable with flux rates in metaphase II oocytes. Further experiments were undertaken to determine the kinetics of cycloleucine uptake into germinal vesicle and metaphase II oocytes. The results obtained are shown in fig. 4a. Uptake of cycloleucine showed Michaelis-Menten kinetics for both types of oocyte. The calculated K, and V,,, values for germinal vesicle and metaphase II oocytes were 0.12?0.05 and 0.15?0.05 mM and 6.6kl.l and 10.5+1.5 nmoles cm+ h-l respectively. It is concluded from these results that the K, is not changed but that the V max is increased significantly during oocyte maturation. Assuming an average oocyte diameter of 120 k and a water content of 85% (equal to that determined previously for mouse oocytes by Loewenstein & Cohen [ 161)it is possible to calculate the intracellular concentration of cycloleucine following incubation in media containing different cycloleucine concentrations. This has been carried out for metaphase II oocytes for a wide range of substrate concentrations. The results are plotted as the cell to medium ratio of cycloleucine concentration in fig. 4b. Metaphase II oocytes are able to take up cycloleucine against a considerable (about 25-fold) gradient of concentration, when in-

in oocytes

339

cubated for 2 h in the presence of 0.1 mM cycloleucine. Similar concentration ratios (not shown in fig. 4 b) can be obtained using even lower concentrations of substrate. The ability of the oocyte to concentrate cycloleucine falls as the medium concentration increases and there is no concentration of amino acid at medium concentrations in excess of 8 mM. The ability of the oocyte to concentrate cycloleucine at low substrate concentrations suggests that this, and possibly other, amino acids enter the oocyte through an energy-requiring process. Cycloleucine oocytes

uptake into mouse

Determinations of amino acid fluxes in mouse oocytes, chosen because of their widespread use in studies on meiotic maturation, were made to provide data against which the rate of amino acid uptake in the sheep oocytes could be compared. Metaphase II oocytes were obtained from mice and incubated for 20 min in concentrations of cycloleucine ranging from 0.05 to 0.4 mM. K, and V,,, values for metaphase II oocytes from mice were 0.25?0.05 mM and 5.79kO.62 nmoles crne2 h-l respectively. While the affinity with which cycloleucine is transported by oocytes from both sheep and mice is similar, the velocity of transport in sheep oocytes is almost double that found in the mouse.

DISCUSSION Gonadotrophin administered in vivo or in vitro significantly increases the rate of amino acid uptake in amphibian oocytes [17]. The increased flux rate in these species is due to an increase in the affinity of the carrier proteins for their specific nutrient (K,) rather than to a change in the EXRCeNRes 119(1979)

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Moor and Smith

maximal velocity of transport across the membrane [18]. From the data of Cross & Brinster [9] it appeared probable that amino acid uptake in mammalian oocytes was similarly increased after exposure to gonadotrophins. However, since the mouse oocytes used in those experiments were all incubated under identical conditions, namely in 1.7 mM [14C]leucine for 60 min, no information on the dynamic aspects of the transport system could be derived from those results. The present experiments, undertaken to obtain this missing information, show that the mechanisms responsible for increased uptake in mammalian oocytes are entirely different from those seen in amphibia. An increase in the maximal velocity of uptake (V,,,) and not in affinity explains the increased rate of flux observed in sheep oocytes during meiotic maturation. Our results, while in close agreement with those of Cross & Brinster [9] on the increase in amino acid transport during maturation, differ with respect to the effect of follicle cells on flux rates. In the experiments on the mouse it was found that follicle cells enhanced amino acid uptake during the germinal vesicle stage, depressed uptake at the first meiotic division and were without effect on metaphase II oocytes [9]. The present experiment using sheep oocytes showed the reverse: amino acid uptake was unaffected by the presence of follicle cells in the germinal vesicle stage but was marginally increased in oocytes at the second meiotic metaphase. It is possible that species variability accounts for the observed differences. Alternatively, the differences may result either from the different amino acids used in the two studies or from the methods used to denude oocytes of their follicle cells. Hyaluronidase was used to remove follicle cells from mouse oocytes. We wished to avoid using enzymes and deExp Cd Res 119 (1979)

nuded the oocytes instead by mechanical means. The action of follicle cells on amino acid uptake in amphibian oocytes is much clearer than in mammals; for HCG stimulation of uptake in amphibia at least one layer of follicle cells is essential [ 17, 181. The mechanism by which follicle cells around amphibian oocytes exert their effects is, however, unclear. Further work on the interactions between follicle cells and oocyte in both mammals and amphibia is clearly warranted. The transport of amino acids by unstimulated follicles in the germinal vesicle stage occurs at a remarkably uniform rate despite considerable differences in the intrafollicular environment to which they were exposed. The three classes of follicles used as sources of germinal vesicle oocytes differed widely with respect to their structure, permeability to macromolecules, receptor development and steroid synthetic capacity [ll, 191. Oestradiol-17P is the principal steroid secreted by follicles in the large nonatretic group, small non-atretic follicles secreted androgens, while both androgen and progesterone are produced by atretic follicles. The uniformity in amino acid fluxes in oocytes obtained from these markedly different hormonal environments suggests that steroids do not act as regulators of amino acid flux in ovine oocytes. This proposition is now being tested more directly in vitro by altering the intrafollicular steroid environment and thereafter measuring rates of uptake by the oocyte. Preliminary results obtained both by supplementation and inhibition of follicular steroid secretion support the concept that steroids do not regulate amino acid transport in mammalian oocytes [20]. These findings are in accord with recent observations in amphibian oocytes where it was found that progesterone and testosterone do not have

Amino acidfluxes in oocytes the same marked effect on amino acid uptake as does HCG [18]. A substantial number of reports have been published on the changes that occur in the egg membrane especially during the preimplantation stages of development [21,22]. In some of these studies attempts have been made to relate the membrane changes with various biosynthetic events taking place within the developing embryo. Thus, in echinoderm eggs hyperpolarization, induced by increased permeability to K ions, occurs shortly after fertilization and this has been implicated in the regulation of protein synthesis [23, 241. In the amphibian oocyte on the other hand no correlation was found between the rate of amino acid uptake and the intracellular events associated with maturation [18]. Our present results show that during normal maturation the increased uptake of amino acids in sheep oocytes coincides with a period of marked change both in the nucleus and in protein synthetic activity [25]. It is, however, equally apparent that amino acid uptake in sheep oocytes can be increased experimentally (by the administration of PMSG in vivo) without inducing accompanying changes in the nucleus or in the overall pattern of protein synthesis. These observations clearly do not diminish the probability that changes in the membrane are nevertheless associated with subtle metabolic events that are essential for full maturation and subsequent normal fertilization and differentiation of the oocyte.

341

REFERENCES 1. Szollosi, D, Oogenesis (ed J D Biggers & A W Schuetz) p. 47. Universitv Park Press, Baltimore, Md (1972)‘. Rodman, T C & Bachvarova, R, J cell biol 70 (1976) 251. Globus, M S & Stein, M P, J exp zoo1 198 (1976) 337. Van Blerkom, J & McGaughey, R W, Dev bio163 (1978) 139. Zamboni, L, Oogenesis (ed J D Bigaers & A W Schuetz) p. 5. University Park Press, Baltimore, Md (1972). 6. Anderson, E & Albertini, D F, J cell biol71 (1974) 680. 7. Cross, M H, Cross, P C & Brinster, R L, Dev biol 33 (1973) 412. 8. Powers, R D & Tupper, J T, Dev biol 38 (1974) 320. 9. Cross, P C & Brinster, R L, Exp cell res 86 (1974) 43. 10. Trounson, A 0, Willadsen, S M & Moor, R M, J agric sci 86 (1976) 609. 11. Moor, R M, Hay, M F, Dott, H M & Cran, D G, J endocrinol77 (1978) 309. 12. Donahue, R P, J exp zoo1 180 (1972) 305. 13. O’Farrel, P H, J biol them 250 (1975) 4007. 14. Austin, C R & Lovack, J E, Exp cell res 15 (1958) 260. 15. Bliss, C I &James, A T, Biometrics 22 (1966) 573. 16. Loewenstein, J D & Cohen, A I, J embryo1 exp morph01 12 (1964) 113. 17. Hallberg, R L & Smith, 0, Dev bio148 (1976) 308. 18 Otero, C, Bravo, R, Rodriguez, C, Paz, B & Allende, J E, Dev bio163 (1978) 213. 19. Hay, M F & Moor, R M, Control of ovulation (ed D B Crighton, N B Haynes, G R Foxcroft & G E Lamming) p. 177. Butterworths, London (1978). 20. Moor, R M & Smith, M W, J physio1284 (1978) 68. 21. Borland, R M, Development in mammals (ed M H Johnson) vol. 1, p. 31. Elsevier, Amsterdam (1977). 22. Biggers, J D, Borland, R M & Powers, R D, The freezing of mammalian embryos, Ciba foundation symp. 52, p. 129. Elsevier, Excerpta medica, Amsterdam (1977). 23. Tupper, J T, Dev bio132 (1973) 140. 24. -Ibid 38 (1974) 332. 25. Wames, G M, Moor, R M & Johnson, M H, J reprod fert 49 (1977) 331. Received August 17, 1978 Revised version received October 30, 1978 Accepted October 3 1, 1978

Exp Cell Res 119 (1979)