- Email: [email protected]
Ultrastructural and cytochemical comparison between calf and cow oocytes
ELSEVIER ULTRASTRUCTURAL AND CYTOCHEMICAL COMPARISON BETWEEN CALF AND COW OOCYTES P. de Paz, 1 A. J. S~.nchez,1 J. De la Fuente,3 C. A. Chamorro, 1 M. Alvarez,2 E. Anel2 and L. Anel2 1Department of Cell Biology and Anatomy, 2Department of Animal Pathology (Reproduction) Faculty of Veterinary, Le6n University, Le6n, Spain, 3Department of Animal Production CIT-INIA, Madrid, Spain Received for publication: March 7, 2000 Accepted: September 20, 2000 ABSTRACT The use of prepubertal females (calves) to obtain oocytes for in vitro fertilization (IVF) programs, is being analyzed currently. This will increase the availability of female oocytes and will allow a reduction of the interval between generations. Differentials in the development capability of calf and cow oocytes have been assessed by different authors, establishing several ultrastructural and metabolic differences between them. This paper analyzes the morphometric and cytochemical differences between calf and cow oocytes through microscopic techniques. The oocytes morphologically classified as good are processed for electron microscopy a) in Epon 812 epoxy resin for morphometric analysis or b) in low temperature Lowycril K4M resin for cytochemical evaluation using Con A, GS, LPA, UEA, and WGA lectins marked with colloidal gold as probes. Calf oocytes show a greater density of microvilli on their surface and a greater number of endocytosic vesicles than those of the cow. On the other hand, cow oocytes show a larger superior mitochondrial population. In the cumulus cells it can be seen that calf oocytes have a greater volume of lipid droplets. Cytochemical analysis shows that calf oocytes have lectin marking restricted to the plasmic membrane, highlighting the presence of LPA. In cow oocytes, lectin marking can be seen both on the plasmic membrane and in the vacuoles, in both cases, with the LPA highlighted. In the zona pellucida of calf and cow oocytes, the same sugars appear (GS, LPA, WGA), and marking with LPA is more extensive in cow oocytes. © 2001 by Elsevter Science Inc
Key words: cow, calf, carbohydrates, lectlns, morphometry, oocyte
INTRODUCTION Several groups have tested the use of prepubertal females (calves) to obtain bovine embryos through maturation and fertilization of oocytes in vitro. The aim of this approach is to take advantage of the greater number of growing follicles in 3 to 4 month old calves (7). Similarly, the
Acknowledgments This work was supported by a grant from CIT INIA (SC94-120) a Correspondence and reprint request: P. de Paz 1 (e-mail address: [email protected]) Thertogenology 55 1107-1116, 2001 © 2001 Elsevter Sctence Inc.
0093-691X/O1/$-see front matter PII: S0093-691X(01)00470-8
Theriogenology
1108
use of young donors in embryo transfer programs offers considerable potential for accelerated gain in domestic livestock through a reduced generation interval (1). Revel et al. (16) proved that the developmental capability of calf oocytes up to blastocysts is significantly lower than that of cow oocytes. This difference is molecularly interpreted on the basis of different hormonal levels in both female groups which determine some cell microenvironments in the follicles. This difference has been assessed by Mermillod et al. (14) through nuclear transfer reaching the conclusion that cow oocytes show some deficiency at a cytoplasmic level. Thus, a significant delay in the migration of cytoplasmic organelles during in-vitro maturation of calf oocytes is described which is not seen in cow oocytes. The data collected by different authors show that differences in oocyte protein secretions may be one factor explaining the difference in the development capability related to age. Biochemical evaluation shows that cytoplasmic maturation is incomplete m calf oocytes (3). These calf oocytes show significantly lower levels of histone H-1 and mitogen-activated protein kxnase than those of the cow relating to these two specific quinase proteins w~th initiation and completion of meiosis. Several variables of oocyte metabolism during IVM also point to significant differences between calves and cows. For instance, radiolabelled amino acid incorporation into proteins synthesised during IVM of calf oocytes decreased after 6 h in culture, whereas a significant decrease in cow oocytes was visible only during the 21 to 24 h period of culture (cow: 7). Further, cow oocytes in the growth phase show a complex series of specific changes in their cytoplasmic and nuclear configuration. These changes are necessary so that the oocyte can reach both its meiotic maturation and its competence for development. The acquisition of meiotic maturation is correlated to follicle and oocyte stze and the morphology of the latter. Cow oocytes derwed from 1 to 2 mm follicles have a significantly lower competence to reach in vitro maturation, being unable to develop beyond the 8-cell stage. Oocytes from 2- to 8 mm follicles have a remarkable capacity of development in vitro up to blastocysts. The same relationship between follicle size and potential development of the oocytes submitted to maturation and fertilization in vitro was studied by Kubota & Yang (10). These authors, through the use of nuclear transfer, established that the poor developmental competence of oocytes derived from very small follicles is attributable to the lack of competence of their cytoplasm. Hence, it has been established that the oocyte size may influence its meiotic competence and it seems necessary for the oocyte to reach at least 80% of its volume to reiniuate its meiosis (11). In this study, several ultrastructural and cytochemical characteristics of the calf and cow oocytes are described and compared so as to analyze their different development capability in FIV programs. MATERIAL AND METHODS Animals Transvaginal and laparoscopic follicular aspiration was performed on four prepubertal heifer calves (3 to 5 months) and four adult cows.
Theriogenology
1109
Calves. The animals were treated previously with a single dose of eCG (1000 IU im). A laparoscopic follicular aspiration was performed 60 h afterwards, under general anesthesia (xylazine: 0.1 mg/kg mi; ketamine: 3 mg/kg iv; isoflurane: 1.5%). The follicles (5 to 15 ram) were aspirated with a device consisting of an 18-gauge needle connected to a tube system with an internal diameter of 1 mm and a vacuum line (flow rate 20 mL./min). For oocyte recovery, TCM199 (Sigma Chemical Co., Madrid, Spain) supplemented with Gentamicine (Sigma Chemical Co.), HEPES (Sigma) and Heparin (Sigma) were used. Cows. The females were treated with a single dose of eCG (2000 IU im) for ovarian superstimulation. After 72 h, the cows were sedated (xylazine: 0.05 mg/kg iv), and epidural anesthesia was performed (5 mL, 0.75% bupivacaine hydrochloride). The ovarian follicles were examined using an ultrasound machine (Aloka SSD500, Tokio, Japan) with a 5 MHz transducer placed inside a vaginal probe with a dorsally-mounted needle guide. The follicles were aspirated (5 to 15 mm) by the same method as the calf follicles. Cumulus oocyte complexes (COCs) were evaluated with a xl0 stereomicroscope. Only good-to-excellent COCs (several complete layers of granulosa cells and oocyte with a homogeneous cytoplasm) were used. Forty-two calf COCs and forty-two cow COCs were obtained. Microscopic Procedures The COCs were classified morphologically and were fixed in 2.5% glutaraldehyde in cacodylate buffer for 45 minutes at room temperature, rinsed in cacodylate buffer and postfixed in 1% osmium tetraoxide in a cacodylate tampon for 60 minutes at room temperature. Samples were stained in block with 1% uranyle acetate, dehydrated in a graded ethanol growing series and were gradually embedded in EMbed 812epoxi resin (EMS, Fort Washington, PA, USA). Blocks were polymerized in an oven at 60QC for 72 h and semi-thin (stained with blue toluidine) and ultra-thin sections (contrasted with uranyl acetate and lead citrate) were obtained. Morphometry To obtain quantitative information on the "average" specimen, unbiased sampling is necessary (20). Twenty-eight calf cumulus oocyte complexes and thirty-three cow complexes collected by follicular aspiration were used. The complexes were processed individuallyfor electron microscopy, yielding 28 and 33 embedded blocks. Finally, 15 blocks of each group of animals were selected at random from each pool to obtain four grids with a ribbon of ultra-thin sections from each block. Ultrastructural study with a transmission electron microscope (TEM) was conducted by random sampling on the TEM screen and by obtaining five micrographs of each grid. The study was conducted at two levels. One was at a magnification of x 5,300 (level I) to analyze cytoplasmic organelles (Numerical density -Nv- and Volume density -Vv-) and the distribution of microvilli in plasma membrane. The other was at a magnification of x 10,000 (Level II) for quantitative evaluation of endocytosis vesicles applying the same sampling model to obtain five micrographs of each grid. The morphometric study of the different organelles was performed on electron micrographs (x 5,300) of each animal group by a semiautomatic system of image analysis (Vidas, Kontron, Germany). Density (structures//.tm of plasma membrane) of the microvilli and the endocytosis
1 110
Theriogenology
vesicles was determined on the respective micrographs. This was achieved by counting the frequencies of the structures per unit length of plasma membrane. Cytochemical Techniques Table I lists the five lectins used in this study, their acronyms and their major sugar specificities. Fourteen calf good oocytes and fifteen cow good oocytes collected by follicular aspiration were used. Cumulus oocyte complexes were fixed in a Kamosky solution (2% formaldehyde and 0.25% glutaraldehyde in Sorensen tampon 0.01 M with 0.15 M of NaCI) for 1 h at 4°C. After fixation, the aldehyde groups were blocked incubating the oocytes in a 0.5 M solution of amonium chloride in PBS 0.1 M for 30 minutes. Dehydration was accomplised by passing the specimen through a graded series of ethanol in water (30, 50, 70, 90 and 100%) at decreasing temperatures (0°, -20°, -25°, -25° and -25°C respectively). After dehydration, specimens were embedded in Lowicryl K4M resin (Polysciences Europe, Eppelheim, Germany) at -25°C. Polymerization was conducted in an ultraviolet light camera at -25°C for 24 h and thereafter at room temperature for 72 h. The ultra-thin sections (see microscopic procedure) were mounted on nickel grids coated with formwar film (0.25%, EMS). The grids then were incubated with a 50% (v/v) solution of each gold-labelled lectins (EY Laboratories, San Mateo, CA, USA) in PBS containing 0.5% BSA (Sigma) and 0.05% TWEEN 20 (Sigma) for 3 hours at room temperature in a humidified atmosphere. The sections were washed three times in distilled water and stained for 5 rain with satured uranyl acetate and lead citrate. The specificity of the lectins was assessed through the coupling of the lectin-gold complex with the appropriate sugar inhibitor in a 0.2 M concentration m PBS for 30 minutes, before the incubation with the respective samples. A sampling method similar to that described for Level I (x 5,300) for morphometry was adopted yielding a set of micrographs for each group of ammals. These micrographs were evaluated semi-quantitativelyas weak, moderate or strong intensities according to the apparent density of gold particles on cellular structures. Statistical Analysis For each group the mean and standard deviation of each morphometric parameter was calculated. Calf and cow data were compared by means of the Student's t-test using Statistica program (Version 5.1, StatSoft). RESULTS Cytochemistry of Lectins (Table 1) Cytochemical data show qualitative differences in various compartments of cow and calf oocytes (Figure lb). These differences were not be seen on the zona pellucida where the binding pattern was similar in cow and calf. Calf oocytes exhibit a lectin-bindingpattern expressed only in the plasma membrane: GS-I (+), LPA (+ +) and WGA (+). However, cow oocytes show a more complex lectin pattern: GS-I
Theriogenology
1111
(+), LPA ( + + ) , U E A ( + ) and W G A (+) for the plasma membrane, and GS-I (+) and LPA (+ + + ) in vacuoles (Figure l d). Morphometry (Tables 2 and 3) The linear density of microvilli on cow oocyte membrane is significantly different from those on calf oocyte (2.5 microvilli/micron versus 1.8 microvilli/micron, respectively). There are also significant differences in the distribution of endocytosis vesicles (calf: 1.8 vesicles/micron, cow: 0.8 vesicles/micron) (Figure la and lc).
Table 1. Cytochemical data for five colloidal gold-lectins in various structures of calf and cow oocytes: the zona pellucida (ZP), the plasma membrane (PM) and the vacuoles (VA) Calf Cow Lectin a Sugar specification ZP PM VA ZP PM VA Con A ct-Manose + GS-I ~-Galactose + + + + LPA N acetylgalactosamme + +++ + ++ +++ UEA a-Fucose + WGA N acetyl~lucosamine + + + + = without marking, + = weak, + + = moderate, + + + = strong a Con A: Concanavalia ensiformis; GS-I: Griffonia simplicifolia; LPA: Limulus polvphemus; UEA: Ulex europaeus, WGA: Triticum vulgare. -
Table 2. Morphometric data for various cytoplasmic compartments of calf and cow oocytes [Vv: Volume density expressed in lam3/lam3 of cytoplasm; Nv: Numerical density expressed in organelles/lam 3 of cytoplasm, (Mean _+SD)]
Calf Cow
Mitochondria Vv Nv 0.040"(0.009) 0.57 (0.18) 0.072b(0.014) 0.68 (0.14)
Oocyte Liptd droplets Vv Nv 0.302(0.100) 0.27 (0.08) 0.286 (0.105) 0.28 (0.05)
Cortical granules Vv Nv 0.010 (0.003) 0.46 (0.19) 0.010 (0.002) 0.40 (0.16)
a, b Columns with different superscripts differ P < 0.05
Table 3. Morphometric data for various cellular compartments of oocyte and the cumulus cells of calf and cow [Vv: Volume density expressed in ~m3/~m 3 of cytoplasm, (Mean _+SD)] Oocyte Microvilh//.tm Endocytosls veslcles/p.m Calf 2.5 a (0.70) 1.5" (0.32) Cow 1.8 b(0.60) 0.8 b(0.11) a, b Columns with different superscripts differ P < 0.05
Cumulus cells Lipid droplets (Vv) 0.039" (0.009) 0.024 b(0.009)
1112
Theriogenology
The mitochondrial population shows significant differences in the volume they occupied within the cell. Cow oocytes show a greater volume of mitochondria (at 7.2% of cytoplasmic volume) than those of the calf (at 4.0%) although the average volume of each mitochondrial unit is lower in cows as can be deduced from the numerical density. Density volume of cortical granules and lipid droplets are not different between calf and cow oocytes. Lipid droplets represent 30% and 29% respectively of cytoplasmic volume, and the cortical granules occupy 1% in both cases. Finally, density volume of lipid droplets of the cumulus cells is significantly more abundant in the cells of calf oocyte (3.9%) than in those from cows (2.4%). Cytochemical and ultrastructural characteristics of calf and cow oocytes are resumed in Figure 2.
Figure 1. a) Endocytotic vesicles in plasma membrane of calf oocyte (arrow) (x 30,000). b) Detail of the gold particles-LPA lectin on the plasma membrane of cow oocyte (x 14,000). c) Microvilli on plasma membrane of calf oocytes (x 3,000). d) Distribution of sugar residues labelled with colloidal gold-LPA lectin in vacuoles of cow oocytes (x 30,000).
Theriogenology
1113
Figure 2. Schematic drawing showing the principal cytochemical and ultrastructural characteristics of calf and cow oocytes indicating the most sigmficant differences found in this study. DISCUSSION The status of the cumulus oophorus IS a criterion determining the quality in the morphological evaluation of oocytes, given the physiological effects of cumulus-oocyte interrelation. Cumulus cells communicate with the oocytes via the gap junctions allowing for the transport of metabolites. These cell-to-cell communications are thought to be important for oocyte maturation (13). Hence, the major cytoplasmic volume of lipid droplets in calf cumulus cells is interpreted in connection with greater density of microvilli on the surface of the oocyte and also with greater development of the endocytosis vesicle complex. These plasma membrane structures are cell mechanisms whereby substances are brought into the cell. It is possible that metabolic exchange between cumulus cells and oocyte may be more active in prepubertal animals than in mature cows and this could be reflected in the ultrastructural characteristics mentioned above. In this sense, calf oocyte showed a major development of endocytosis structures and calf cumulus cells showed more lipid droplets. Hashimoto et al. (8) suggest that cumulus cells benefit bovine oocyte development either by secreting soluble factors that induce developmental competence or by removing an embryo development-suppressive component from its medium. On the other hand, it was shown that follicle cells secrete substances that induce germinal vesicle breakdown in mouse oocytes (5). Hill et al. (9) suggest that caveolae (non-clathrin-coated plasma membrane invaginations) and caveolincontaining vesicles are present both in cow cumulus cells and oocytes. These structures may play
1 114
Theriogenology
important roles in signal transduction processes that are crucial to oocyte maturation (9). A morphological classification of the cellular coupling in bovine cumulus oocyte complexes was identified showing that oocytes from antral follicles in the cow form a heterogeneous population (4). De Loos et al. (4) identified four groups of COCs that showed differences in morphological characteristics that were partially reflected in the functionality of the intercellular coupling. Similarly, Suzuki et al. (19) observe that a brief heat shock changes the bovine oocyte vitellin surface from a well-developed microvilli-predominantpattern to a small-cytoplasmic-protrusions predominant pattern, and that the effect is apparently earlier and in a higher proportion in aged than in young oocytes. This suggests the existence of a different capability between young and mature oocytes to develop membrane structures. Oocytes of prepubertal calf are less developmentally competent than adult cow oocytes. Although these oocytes are able to mature, be fertilized and cleave at normal rates, their development to blastocyst stage is reduced. Several types of evidence show that the ability of these oocytes to develop depends on their hormonal environment and is associated with sequential changes in puberty (16). In this sense, Earl et al. (6) indicate that although calf oocytes may exhibit slightly reduced rates of development to blastocyst in vitro, such blastocysts are able to establish full-term pregnancy rates essentially similar to those expected from in-vitro produced embryos from adult cows and not much lower than in-vivo embryos recovered by uterine flushing of superovulated cows. This low developmental competence of prepubertal oocytes could be explained by a deficiency at a cytoplasmic level (14). One possible explanation for this cytoplasmic immaturity is given by Crozet et al. (2) through analysis of the developmental competence of goat oocytes from follicles of different size categories. Their results indicate that the maternal mRNAs needed for compaction and for blastocyst differentiation are accumulated in the oocyte during the final phase of follicular growth and, therefore, they are not present in small follicles. One bit of morphological evidence of this cytoplasmic maturation may be the redistribution of cortical granulles in the oocyte cytoplasm (12). As oocytes matured the distribution of these granules in bovine oocytes varied, beginning with a clustered pattern that became more uniformly distributed. In this way our results indicate that there are no quantitative differences between cortical granules and between lipid droplets in prepubertal and pubertal oocytes. However, the mitochondrial population is different. Cow oocytes show a more abundant mitochondrial population than do calf oocytes. This fact suggests that the energy metabolism of cow oocytes is greater than in calf oocytes. Rieger et al. (17) showed that the relative effects of some cellular growth factors on oocyte piruvate metabolism generally parelleled their effects on cleavage and subsequent development, suggesting that mitochondrial function is related to developmental potential. The distribution pattern of carbohydrates in the zona pellucida does not show differences between calf and cow. In agreement with a previous study (15) our results showed that the cow zona pellucida presented B-galactose, N-acetylgalactosamine and N- acetylglucosamine, although our quantitative evaluation is different. This discrepancy might reflect the difference in the histochemical approach used. The gold colloidal lectin labeling applied in the present study is undoubtely more reliable for localization of lectin receptor sites. Skutelsky et al. (18) demonstrated
Theriogenology
1115
that B-galactose and N-acetylglucosamine are commonly present in most mammalian zonae pellucidae. The differences in chemical composmon of the zona pellucida in immature oocytes from superovulated and unstimulated cows were evaluated by Parilio et al. (15). Treatment with endogenous gonadotropins did not induce changes in the glycoconjugate composition of the zona pellucida. The similarity in carbohydrate expression between calf and cow zona pellucida reported in the present study may reflect that the changes in the hormonal enwronment during sexual maturation do not change the sugar residue pattern expressed on the zona pellucida. Variations in the distribution of sugar residues in plasma membrane and vacuoles observed between calf and cow oocytes suggest differences in the physiological and biochemical characteristics of cow and calf oocytes during oocyte maturation (3). According to Damiani et al. (3) ooplasmic maturation is incomplete in oocytes from prepuberal heifers and calf oocytes had only 50% of the relative kinase activity of cow oocytes after 24 h of maturation. In conclusion, our results suggest that the ultrastructural characteristics of calf oocytes probably reflect a greater activity of metabolic exchange with the cumulus cells. However, cow oocytes show a more abundant mitochondrial population suggesting that oradatwe metabolism increases during oocyte maturation. REFERENCES 1. Armstrong DT, Kotaras PJ, Earl CR. Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb. Reprod Fertil Dev 1997; 9: 333-329. 2. Crozet N, Ahmed-Ali M, Dubos MP. Development competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J Reprod Fertil 1995; 103: 293-298. 3. Damiani P, Fissore RA, Cibelli JB, Robl JM, Duby RT. Evaluation of cytoplasmic maturation of calf oocytes. Theriogenology 1998; 49:310. 4. De Loos F, Kastrop P, Van Maurik P, Van Beneden T, Kruip AM. Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes. Mol Reprod Dev 1991; 28: 255259. 5. Downs SM. The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocytes. Dev Biol 1995; 167: 502-512. 6. Earl CR, Fry RC, Maclellan LJ, Kelly JM, Armstrong DT. In vitro fertilization and developmental potential prepubertal calf oocytes. In: Lauria A, Gandolfi F, Enne G, Gianardi L (Eds), Gametes: Development and Function. Roma. Serono Symposia. 1998; 115137. 7. Gandolfi F, Milanesi E, Pocar P, Luciano AM, Brevini TAL, Acocella F, Lauria A, Armstrong DT. Comparative analysis of calf and cow oocytes during in vitro maturation. Mol Reprod Dev 1998; 49: 168-175. 8. Hashimoto S, Saeki K, Nagao Y, Minami N, Yamada M, Utsumi K. Effects of cumulus cell density during in vitro maturation on the developmental competence of bowne oocytes. Theriogenology 1998; 49:1451-1463. 9. Hill JL, Fissore RA, Duby RT, Gross DJ. Occurrence and distribution of caveolin in mouse and bovine oocytes. Theriogenology 1998; 49:181.
1116
Theriogenology
10. Kubota C, Yang X. Cytoplasmic incompetence results in poor development of bovine oocytes derived from small follicles. Theriogenology 1998; 49:183. 11. Ledda S, Bogliolo L, Leoni G, Loi P, Cappal P, Naitana S. Meiotic and development competence of in vitro matured lamb oocytes. In: Lauria A, Gandolfi F, Enne G, Gianardi L (Eds), Gametes: Development and Function. Roma. Serono Symposia. 1998; 101-114 12. Long CR, Damiani P, Pinto-Correia, MacLean RA, Duby RT, Robl JM. Morphology and subsequent development in culture of bovine oocytes matured in vitro under various conditions of fertilization. J Reprod Fertil 1994; 102: 361-369. 13. Mattioli M & Barboni B. Induction of oocyte maturation. In: Lauria A, Gandolfi F, Enne G, Gianardi L (Eds), Gametes: Development and Function. Roma. Serono Symposia. 1998; 141153 14. Mermdlod P, Le Bourhis D, Lonergan P, Khatir H, Heyman Y. Assessment of cytoplasmic competence of prepubertal calf oocytes by use nuclear transfer. Theriogenology 1998; 49: 187. 15. Parillo F, Verim Supplizi A, Stradaioli G, Tortora G, Monaci M. Identificazione istochimica mediante lectine dei glicoconiugati della zona pellucida di ovocellule di bovine sottoposte a tratamento superovulatorio. Acta Med Vet 1994: 40: 215-221. 16. Revel F, Mermillod P, Peynot N, Renard JP, Heyman Y. Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows. J Reprod Fertil 1995; 103: 115-120. 17. Rieger D, Luclano AM, Modina S, Pocar P, Lauria A, Gandolfi F. The effects of epidermal growth factor and insulin-like growth factor I on the metabolic activity, nuclear maturation and subsequent development of cattle oocytes in vitro. J Reprod Fertil 1998; 112: 123-130. 18. Skutelsky E, Ranen E, Shalgi R. Variations in the distribution of sugar residues in the zona pelluclda as possible species-specific determinants of mammalian oocytes. J Reprod Fertil 1994; 100: 35-41. 19. Suzuki H, Ju JC, Parks JE, Yang X, Effect of heat shock on the surface ultrastructural characteristics of the bovine oocytes. Theriogenology 1998; 49: 239. 20. Williams M (1977) Stereological techniques. In AM Glauert (ed): Practical methods in electron microscopy, Vol. 6, part II. Amsterdam: North Holland/American Elsevier.
Key words: cow, calf, carbohydrates, lectlns, morphometry, oocyte
INTRODUCTION Several groups have tested the use of prepubertal females (calves) to obtain bovine embryos through maturation and fertilization of oocytes in vitro. The aim of this approach is to take advantage of the greater number of growing follicles in 3 to 4 month old calves (7). Similarly, the
Acknowledgments This work was supported by a grant from CIT INIA (SC94-120) a Correspondence and reprint request: P. de Paz 1 (e-mail address: [email protected]) Thertogenology 55 1107-1116, 2001 © 2001 Elsevter Sctence Inc.
0093-691X/O1/$-see front matter PII: S0093-691X(01)00470-8
Theriogenology
1108
use of young donors in embryo transfer programs offers considerable potential for accelerated gain in domestic livestock through a reduced generation interval (1). Revel et al. (16) proved that the developmental capability of calf oocytes up to blastocysts is significantly lower than that of cow oocytes. This difference is molecularly interpreted on the basis of different hormonal levels in both female groups which determine some cell microenvironments in the follicles. This difference has been assessed by Mermillod et al. (14) through nuclear transfer reaching the conclusion that cow oocytes show some deficiency at a cytoplasmic level. Thus, a significant delay in the migration of cytoplasmic organelles during in-vitro maturation of calf oocytes is described which is not seen in cow oocytes. The data collected by different authors show that differences in oocyte protein secretions may be one factor explaining the difference in the development capability related to age. Biochemical evaluation shows that cytoplasmic maturation is incomplete m calf oocytes (3). These calf oocytes show significantly lower levels of histone H-1 and mitogen-activated protein kxnase than those of the cow relating to these two specific quinase proteins w~th initiation and completion of meiosis. Several variables of oocyte metabolism during IVM also point to significant differences between calves and cows. For instance, radiolabelled amino acid incorporation into proteins synthesised during IVM of calf oocytes decreased after 6 h in culture, whereas a significant decrease in cow oocytes was visible only during the 21 to 24 h period of culture (cow: 7). Further, cow oocytes in the growth phase show a complex series of specific changes in their cytoplasmic and nuclear configuration. These changes are necessary so that the oocyte can reach both its meiotic maturation and its competence for development. The acquisition of meiotic maturation is correlated to follicle and oocyte stze and the morphology of the latter. Cow oocytes derwed from 1 to 2 mm follicles have a significantly lower competence to reach in vitro maturation, being unable to develop beyond the 8-cell stage. Oocytes from 2- to 8 mm follicles have a remarkable capacity of development in vitro up to blastocysts. The same relationship between follicle size and potential development of the oocytes submitted to maturation and fertilization in vitro was studied by Kubota & Yang (10). These authors, through the use of nuclear transfer, established that the poor developmental competence of oocytes derived from very small follicles is attributable to the lack of competence of their cytoplasm. Hence, it has been established that the oocyte size may influence its meiotic competence and it seems necessary for the oocyte to reach at least 80% of its volume to reiniuate its meiosis (11). In this study, several ultrastructural and cytochemical characteristics of the calf and cow oocytes are described and compared so as to analyze their different development capability in FIV programs. MATERIAL AND METHODS Animals Transvaginal and laparoscopic follicular aspiration was performed on four prepubertal heifer calves (3 to 5 months) and four adult cows.
Theriogenology
1109
Calves. The animals were treated previously with a single dose of eCG (1000 IU im). A laparoscopic follicular aspiration was performed 60 h afterwards, under general anesthesia (xylazine: 0.1 mg/kg mi; ketamine: 3 mg/kg iv; isoflurane: 1.5%). The follicles (5 to 15 ram) were aspirated with a device consisting of an 18-gauge needle connected to a tube system with an internal diameter of 1 mm and a vacuum line (flow rate 20 mL./min). For oocyte recovery, TCM199 (Sigma Chemical Co., Madrid, Spain) supplemented with Gentamicine (Sigma Chemical Co.), HEPES (Sigma) and Heparin (Sigma) were used. Cows. The females were treated with a single dose of eCG (2000 IU im) for ovarian superstimulation. After 72 h, the cows were sedated (xylazine: 0.05 mg/kg iv), and epidural anesthesia was performed (5 mL, 0.75% bupivacaine hydrochloride). The ovarian follicles were examined using an ultrasound machine (Aloka SSD500, Tokio, Japan) with a 5 MHz transducer placed inside a vaginal probe with a dorsally-mounted needle guide. The follicles were aspirated (5 to 15 mm) by the same method as the calf follicles. Cumulus oocyte complexes (COCs) were evaluated with a xl0 stereomicroscope. Only good-to-excellent COCs (several complete layers of granulosa cells and oocyte with a homogeneous cytoplasm) were used. Forty-two calf COCs and forty-two cow COCs were obtained. Microscopic Procedures The COCs were classified morphologically and were fixed in 2.5% glutaraldehyde in cacodylate buffer for 45 minutes at room temperature, rinsed in cacodylate buffer and postfixed in 1% osmium tetraoxide in a cacodylate tampon for 60 minutes at room temperature. Samples were stained in block with 1% uranyle acetate, dehydrated in a graded ethanol growing series and were gradually embedded in EMbed 812epoxi resin (EMS, Fort Washington, PA, USA). Blocks were polymerized in an oven at 60QC for 72 h and semi-thin (stained with blue toluidine) and ultra-thin sections (contrasted with uranyl acetate and lead citrate) were obtained. Morphometry To obtain quantitative information on the "average" specimen, unbiased sampling is necessary (20). Twenty-eight calf cumulus oocyte complexes and thirty-three cow complexes collected by follicular aspiration were used. The complexes were processed individuallyfor electron microscopy, yielding 28 and 33 embedded blocks. Finally, 15 blocks of each group of animals were selected at random from each pool to obtain four grids with a ribbon of ultra-thin sections from each block. Ultrastructural study with a transmission electron microscope (TEM) was conducted by random sampling on the TEM screen and by obtaining five micrographs of each grid. The study was conducted at two levels. One was at a magnification of x 5,300 (level I) to analyze cytoplasmic organelles (Numerical density -Nv- and Volume density -Vv-) and the distribution of microvilli in plasma membrane. The other was at a magnification of x 10,000 (Level II) for quantitative evaluation of endocytosis vesicles applying the same sampling model to obtain five micrographs of each grid. The morphometric study of the different organelles was performed on electron micrographs (x 5,300) of each animal group by a semiautomatic system of image analysis (Vidas, Kontron, Germany). Density (structures//.tm of plasma membrane) of the microvilli and the endocytosis
1 110
Theriogenology
vesicles was determined on the respective micrographs. This was achieved by counting the frequencies of the structures per unit length of plasma membrane. Cytochemical Techniques Table I lists the five lectins used in this study, their acronyms and their major sugar specificities. Fourteen calf good oocytes and fifteen cow good oocytes collected by follicular aspiration were used. Cumulus oocyte complexes were fixed in a Kamosky solution (2% formaldehyde and 0.25% glutaraldehyde in Sorensen tampon 0.01 M with 0.15 M of NaCI) for 1 h at 4°C. After fixation, the aldehyde groups were blocked incubating the oocytes in a 0.5 M solution of amonium chloride in PBS 0.1 M for 30 minutes. Dehydration was accomplised by passing the specimen through a graded series of ethanol in water (30, 50, 70, 90 and 100%) at decreasing temperatures (0°, -20°, -25°, -25° and -25°C respectively). After dehydration, specimens were embedded in Lowicryl K4M resin (Polysciences Europe, Eppelheim, Germany) at -25°C. Polymerization was conducted in an ultraviolet light camera at -25°C for 24 h and thereafter at room temperature for 72 h. The ultra-thin sections (see microscopic procedure) were mounted on nickel grids coated with formwar film (0.25%, EMS). The grids then were incubated with a 50% (v/v) solution of each gold-labelled lectins (EY Laboratories, San Mateo, CA, USA) in PBS containing 0.5% BSA (Sigma) and 0.05% TWEEN 20 (Sigma) for 3 hours at room temperature in a humidified atmosphere. The sections were washed three times in distilled water and stained for 5 rain with satured uranyl acetate and lead citrate. The specificity of the lectins was assessed through the coupling of the lectin-gold complex with the appropriate sugar inhibitor in a 0.2 M concentration m PBS for 30 minutes, before the incubation with the respective samples. A sampling method similar to that described for Level I (x 5,300) for morphometry was adopted yielding a set of micrographs for each group of ammals. These micrographs were evaluated semi-quantitativelyas weak, moderate or strong intensities according to the apparent density of gold particles on cellular structures. Statistical Analysis For each group the mean and standard deviation of each morphometric parameter was calculated. Calf and cow data were compared by means of the Student's t-test using Statistica program (Version 5.1, StatSoft). RESULTS Cytochemistry of Lectins (Table 1) Cytochemical data show qualitative differences in various compartments of cow and calf oocytes (Figure lb). These differences were not be seen on the zona pellucida where the binding pattern was similar in cow and calf. Calf oocytes exhibit a lectin-bindingpattern expressed only in the plasma membrane: GS-I (+), LPA (+ +) and WGA (+). However, cow oocytes show a more complex lectin pattern: GS-I
Theriogenology
1111
(+), LPA ( + + ) , U E A ( + ) and W G A (+) for the plasma membrane, and GS-I (+) and LPA (+ + + ) in vacuoles (Figure l d). Morphometry (Tables 2 and 3) The linear density of microvilli on cow oocyte membrane is significantly different from those on calf oocyte (2.5 microvilli/micron versus 1.8 microvilli/micron, respectively). There are also significant differences in the distribution of endocytosis vesicles (calf: 1.8 vesicles/micron, cow: 0.8 vesicles/micron) (Figure la and lc).
Table 1. Cytochemical data for five colloidal gold-lectins in various structures of calf and cow oocytes: the zona pellucida (ZP), the plasma membrane (PM) and the vacuoles (VA) Calf Cow Lectin a Sugar specification ZP PM VA ZP PM VA Con A ct-Manose + GS-I ~-Galactose + + + + LPA N acetylgalactosamme + +++ + ++ +++ UEA a-Fucose + WGA N acetyl~lucosamine + + + + = without marking, + = weak, + + = moderate, + + + = strong a Con A: Concanavalia ensiformis; GS-I: Griffonia simplicifolia; LPA: Limulus polvphemus; UEA: Ulex europaeus, WGA: Triticum vulgare. -
Table 2. Morphometric data for various cytoplasmic compartments of calf and cow oocytes [Vv: Volume density expressed in lam3/lam3 of cytoplasm; Nv: Numerical density expressed in organelles/lam 3 of cytoplasm, (Mean _+SD)]
Calf Cow
Mitochondria Vv Nv 0.040"(0.009) 0.57 (0.18) 0.072b(0.014) 0.68 (0.14)
Oocyte Liptd droplets Vv Nv 0.302(0.100) 0.27 (0.08) 0.286 (0.105) 0.28 (0.05)
Cortical granules Vv Nv 0.010 (0.003) 0.46 (0.19) 0.010 (0.002) 0.40 (0.16)
a, b Columns with different superscripts differ P < 0.05
Table 3. Morphometric data for various cellular compartments of oocyte and the cumulus cells of calf and cow [Vv: Volume density expressed in ~m3/~m 3 of cytoplasm, (Mean _+SD)] Oocyte Microvilh//.tm Endocytosls veslcles/p.m Calf 2.5 a (0.70) 1.5" (0.32) Cow 1.8 b(0.60) 0.8 b(0.11) a, b Columns with different superscripts differ P < 0.05
Cumulus cells Lipid droplets (Vv) 0.039" (0.009) 0.024 b(0.009)
1112
Theriogenology
The mitochondrial population shows significant differences in the volume they occupied within the cell. Cow oocytes show a greater volume of mitochondria (at 7.2% of cytoplasmic volume) than those of the calf (at 4.0%) although the average volume of each mitochondrial unit is lower in cows as can be deduced from the numerical density. Density volume of cortical granules and lipid droplets are not different between calf and cow oocytes. Lipid droplets represent 30% and 29% respectively of cytoplasmic volume, and the cortical granules occupy 1% in both cases. Finally, density volume of lipid droplets of the cumulus cells is significantly more abundant in the cells of calf oocyte (3.9%) than in those from cows (2.4%). Cytochemical and ultrastructural characteristics of calf and cow oocytes are resumed in Figure 2.
Figure 1. a) Endocytotic vesicles in plasma membrane of calf oocyte (arrow) (x 30,000). b) Detail of the gold particles-LPA lectin on the plasma membrane of cow oocyte (x 14,000). c) Microvilli on plasma membrane of calf oocytes (x 3,000). d) Distribution of sugar residues labelled with colloidal gold-LPA lectin in vacuoles of cow oocytes (x 30,000).
Theriogenology
1113
Figure 2. Schematic drawing showing the principal cytochemical and ultrastructural characteristics of calf and cow oocytes indicating the most sigmficant differences found in this study. DISCUSSION The status of the cumulus oophorus IS a criterion determining the quality in the morphological evaluation of oocytes, given the physiological effects of cumulus-oocyte interrelation. Cumulus cells communicate with the oocytes via the gap junctions allowing for the transport of metabolites. These cell-to-cell communications are thought to be important for oocyte maturation (13). Hence, the major cytoplasmic volume of lipid droplets in calf cumulus cells is interpreted in connection with greater density of microvilli on the surface of the oocyte and also with greater development of the endocytosis vesicle complex. These plasma membrane structures are cell mechanisms whereby substances are brought into the cell. It is possible that metabolic exchange between cumulus cells and oocyte may be more active in prepubertal animals than in mature cows and this could be reflected in the ultrastructural characteristics mentioned above. In this sense, calf oocyte showed a major development of endocytosis structures and calf cumulus cells showed more lipid droplets. Hashimoto et al. (8) suggest that cumulus cells benefit bovine oocyte development either by secreting soluble factors that induce developmental competence or by removing an embryo development-suppressive component from its medium. On the other hand, it was shown that follicle cells secrete substances that induce germinal vesicle breakdown in mouse oocytes (5). Hill et al. (9) suggest that caveolae (non-clathrin-coated plasma membrane invaginations) and caveolincontaining vesicles are present both in cow cumulus cells and oocytes. These structures may play
1 114
Theriogenology
important roles in signal transduction processes that are crucial to oocyte maturation (9). A morphological classification of the cellular coupling in bovine cumulus oocyte complexes was identified showing that oocytes from antral follicles in the cow form a heterogeneous population (4). De Loos et al. (4) identified four groups of COCs that showed differences in morphological characteristics that were partially reflected in the functionality of the intercellular coupling. Similarly, Suzuki et al. (19) observe that a brief heat shock changes the bovine oocyte vitellin surface from a well-developed microvilli-predominantpattern to a small-cytoplasmic-protrusions predominant pattern, and that the effect is apparently earlier and in a higher proportion in aged than in young oocytes. This suggests the existence of a different capability between young and mature oocytes to develop membrane structures. Oocytes of prepubertal calf are less developmentally competent than adult cow oocytes. Although these oocytes are able to mature, be fertilized and cleave at normal rates, their development to blastocyst stage is reduced. Several types of evidence show that the ability of these oocytes to develop depends on their hormonal environment and is associated with sequential changes in puberty (16). In this sense, Earl et al. (6) indicate that although calf oocytes may exhibit slightly reduced rates of development to blastocyst in vitro, such blastocysts are able to establish full-term pregnancy rates essentially similar to those expected from in-vitro produced embryos from adult cows and not much lower than in-vivo embryos recovered by uterine flushing of superovulated cows. This low developmental competence of prepubertal oocytes could be explained by a deficiency at a cytoplasmic level (14). One possible explanation for this cytoplasmic immaturity is given by Crozet et al. (2) through analysis of the developmental competence of goat oocytes from follicles of different size categories. Their results indicate that the maternal mRNAs needed for compaction and for blastocyst differentiation are accumulated in the oocyte during the final phase of follicular growth and, therefore, they are not present in small follicles. One bit of morphological evidence of this cytoplasmic maturation may be the redistribution of cortical granulles in the oocyte cytoplasm (12). As oocytes matured the distribution of these granules in bovine oocytes varied, beginning with a clustered pattern that became more uniformly distributed. In this way our results indicate that there are no quantitative differences between cortical granules and between lipid droplets in prepubertal and pubertal oocytes. However, the mitochondrial population is different. Cow oocytes show a more abundant mitochondrial population than do calf oocytes. This fact suggests that the energy metabolism of cow oocytes is greater than in calf oocytes. Rieger et al. (17) showed that the relative effects of some cellular growth factors on oocyte piruvate metabolism generally parelleled their effects on cleavage and subsequent development, suggesting that mitochondrial function is related to developmental potential. The distribution pattern of carbohydrates in the zona pellucida does not show differences between calf and cow. In agreement with a previous study (15) our results showed that the cow zona pellucida presented B-galactose, N-acetylgalactosamine and N- acetylglucosamine, although our quantitative evaluation is different. This discrepancy might reflect the difference in the histochemical approach used. The gold colloidal lectin labeling applied in the present study is undoubtely more reliable for localization of lectin receptor sites. Skutelsky et al. (18) demonstrated
Theriogenology
1115
that B-galactose and N-acetylglucosamine are commonly present in most mammalian zonae pellucidae. The differences in chemical composmon of the zona pellucida in immature oocytes from superovulated and unstimulated cows were evaluated by Parilio et al. (15). Treatment with endogenous gonadotropins did not induce changes in the glycoconjugate composition of the zona pellucida. The similarity in carbohydrate expression between calf and cow zona pellucida reported in the present study may reflect that the changes in the hormonal enwronment during sexual maturation do not change the sugar residue pattern expressed on the zona pellucida. Variations in the distribution of sugar residues in plasma membrane and vacuoles observed between calf and cow oocytes suggest differences in the physiological and biochemical characteristics of cow and calf oocytes during oocyte maturation (3). According to Damiani et al. (3) ooplasmic maturation is incomplete in oocytes from prepuberal heifers and calf oocytes had only 50% of the relative kinase activity of cow oocytes after 24 h of maturation. In conclusion, our results suggest that the ultrastructural characteristics of calf oocytes probably reflect a greater activity of metabolic exchange with the cumulus cells. However, cow oocytes show a more abundant mitochondrial population suggesting that oradatwe metabolism increases during oocyte maturation. REFERENCES 1. Armstrong DT, Kotaras PJ, Earl CR. Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb. Reprod Fertil Dev 1997; 9: 333-329. 2. Crozet N, Ahmed-Ali M, Dubos MP. Development competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro. J Reprod Fertil 1995; 103: 293-298. 3. Damiani P, Fissore RA, Cibelli JB, Robl JM, Duby RT. Evaluation of cytoplasmic maturation of calf oocytes. Theriogenology 1998; 49:310. 4. De Loos F, Kastrop P, Van Maurik P, Van Beneden T, Kruip AM. Heterologous cell contacts and metabolic coupling in bovine cumulus oocyte complexes. Mol Reprod Dev 1991; 28: 255259. 5. Downs SM. The influence of glucose, cumulus cells, and metabolic coupling on ATP levels and meiotic control in the isolated mouse oocytes. Dev Biol 1995; 167: 502-512. 6. Earl CR, Fry RC, Maclellan LJ, Kelly JM, Armstrong DT. In vitro fertilization and developmental potential prepubertal calf oocytes. In: Lauria A, Gandolfi F, Enne G, Gianardi L (Eds), Gametes: Development and Function. Roma. Serono Symposia. 1998; 115137. 7. Gandolfi F, Milanesi E, Pocar P, Luciano AM, Brevini TAL, Acocella F, Lauria A, Armstrong DT. Comparative analysis of calf and cow oocytes during in vitro maturation. Mol Reprod Dev 1998; 49: 168-175. 8. Hashimoto S, Saeki K, Nagao Y, Minami N, Yamada M, Utsumi K. Effects of cumulus cell density during in vitro maturation on the developmental competence of bowne oocytes. Theriogenology 1998; 49:1451-1463. 9. Hill JL, Fissore RA, Duby RT, Gross DJ. Occurrence and distribution of caveolin in mouse and bovine oocytes. Theriogenology 1998; 49:181.
1116
Theriogenology
10. Kubota C, Yang X. Cytoplasmic incompetence results in poor development of bovine oocytes derived from small follicles. Theriogenology 1998; 49:183. 11. Ledda S, Bogliolo L, Leoni G, Loi P, Cappal P, Naitana S. Meiotic and development competence of in vitro matured lamb oocytes. In: Lauria A, Gandolfi F, Enne G, Gianardi L (Eds), Gametes: Development and Function. Roma. Serono Symposia. 1998; 101-114 12. Long CR, Damiani P, Pinto-Correia, MacLean RA, Duby RT, Robl JM. Morphology and subsequent development in culture of bovine oocytes matured in vitro under various conditions of fertilization. J Reprod Fertil 1994; 102: 361-369. 13. Mattioli M & Barboni B. Induction of oocyte maturation. In: Lauria A, Gandolfi F, Enne G, Gianardi L (Eds), Gametes: Development and Function. Roma. Serono Symposia. 1998; 141153 14. Mermdlod P, Le Bourhis D, Lonergan P, Khatir H, Heyman Y. Assessment of cytoplasmic competence of prepubertal calf oocytes by use nuclear transfer. Theriogenology 1998; 49: 187. 15. Parillo F, Verim Supplizi A, Stradaioli G, Tortora G, Monaci M. Identificazione istochimica mediante lectine dei glicoconiugati della zona pellucida di ovocellule di bovine sottoposte a tratamento superovulatorio. Acta Med Vet 1994: 40: 215-221. 16. Revel F, Mermillod P, Peynot N, Renard JP, Heyman Y. Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows. J Reprod Fertil 1995; 103: 115-120. 17. Rieger D, Luclano AM, Modina S, Pocar P, Lauria A, Gandolfi F. The effects of epidermal growth factor and insulin-like growth factor I on the metabolic activity, nuclear maturation and subsequent development of cattle oocytes in vitro. J Reprod Fertil 1998; 112: 123-130. 18. Skutelsky E, Ranen E, Shalgi R. Variations in the distribution of sugar residues in the zona pelluclda as possible species-specific determinants of mammalian oocytes. J Reprod Fertil 1994; 100: 35-41. 19. Suzuki H, Ju JC, Parks JE, Yang X, Effect of heat shock on the surface ultrastructural characteristics of the bovine oocytes. Theriogenology 1998; 49: 239. 20. Williams M (1977) Stereological techniques. In AM Glauert (ed): Practical methods in electron microscopy, Vol. 6, part II. Amsterdam: North Holland/American Elsevier.