‘ A 1 ‘ ' «a :3? ,3 "3‘! Lin-«w ' mam!» {Me Ufi§V%rauiy THESIS 7*— This is to certify that the thesis entitled BIOCHEMICAL ASPECTS OF EMBRYONIC DEVELOPMENT IN PRIMATES FOLLOWING fl VITRO FERTILIZATION presented by Reinhold J. Hutz has been accepted towards fulfillment of the requirements for ph.D. degreein Physiology w: Mme/2 5.“) Major professor Date April 8, 1983 MSU LIBRARIES m \— RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. BIOCHEMICAL ASPECTS OF EMBRYONIC DEVELOPMENT IN PRIMATES FOLLOWING IN_VITRO FERTILIZATION By Reinhold J. Hutz A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1983 ABSTRACT BIOCHEMICAL ASPECTS OF EMBRYONIC DEVELOPMENT IN PRIMATES FOLLOWING IN_VITRO FERTILIZATION By Reinhold J. Hutz These studies were designed to evaluate the viability and bio- chemical alterations in squirrel monkey ova fertilized and developed 12m. Exclusion of the vital dye trypan blue and uptake of fluorescein diacetate, by hamster and squirrel monkey (in vitrg_fertilized) ova showed high correlations with in_vitrg_development and relative RNA and protein synthesis. 3H-Uridine incorporation in unfertilized squirrel monkey oocytes was diminished with time after HCG administra- 3 tion. There was an increase in H-uridine incorporation after ferti- lization jg_vjtrg, with another rise following the second cleavage division. 3H-Leucine incorporation decreased with oocyte maturation, and then remained constant to the two-cell stage. Uptake of estradiol-l7e and progesterone by squirrel monkey ova increased with fertilization jg_vitrg_from 0.59 to 0.87, and 0.2l to 0.49 picomoles/embryo/Z hr, respectively, and to l.20 and 0.38 at the two-cell stage. Changing the length of FSH treatment prior to HCG- induced ovulation in Saimiri did not affect uptake. However, the PMS-HCG superovulatory regimen reduced uptake of both steroids at all Reinhold J. Hutz embryonic stages in the hamster. There was no effect on 3H-uridine incorporation. Unfertilized, immature oocytes from squirrel monkeys consumed 4.85 nanoliters oxygen/oocyte/4 hr. 14 002 production was 4l.9 pico- gram-atoms glucose carbon incorporated/oocyte/4 hr. The molar ratio of 14CO2 produced from glucose to total oxygen uptake was estimated at 0.l9. Uptake of 2-deoxyglucose by unfertilized oocytes from squirrel monkeys was not affected by the addition of either l0 nM or T uM insulin. There was no change in 2-deoxyglucose uptake at ig_vitrg_ fertilization. Degenerate ova had significantly reduced levels of 2? deoxyglucose uptake. Detectable biochemical changes occur in the primate ovum with in vitrg_fertilization. These include augmented 3H-uridine incorporation and steroid uptake, and diminished incorporation of 3H-leucine. Glu- cose utilization remains quite low in early primate embryos. These results indicate normal metabolic development of primate embryos fertilized jg_vitro that is similar to preimplantation development of embryos in other mammalian species. Trypan blue, fluorescein diace- tate and 2-deoxyglucose are good viability indicators of early primate embryos. May a love limitless in its breadth And knowing no bounds, From God and man, 'Till eternity resound. ii ACKNOWLEDGEMENTS The author wishes to express his thanks and appreciation to an advisor nonpareil, Dr. W. Richard Dukelow, for taking so much time and energy to relate a clear concept of the qualities a good scientist should maintain. A scientist is much more than a cold, calculating experimenter. He can be a warm, philanthropic individual who exer- cises ethical judgement and maintains impeccable rapport with his fellow human beings. Thanks must also go to the members of the author's guidance committee, Drs. Surinder Aggarwal, Lynwood Clemens, Thomas Emerson, Lester Wolterink and Raymond Nachreiner, for their advice and review of this manuscript. The author is grateful for the collaboration of Drs. Maya Ghosh and Gerald Holzman on the steroid and human projects, respectively. The expert technical assistance of our supersecretary, Ms. L. M. "Bonnie" Cleeves, and fellow graduate stu- dents, Phil Chan and Franco DeMayo, is greatly appreciated. Others, without whom the laboratory could not function, are too numerous to mention. A special thank you goes to Ms. Diane Hummel for the final typing of this manuscript. Finally, love and gratitude go out to my future wife, Dr. Irene O'Shaughnessy, and my parents for their support and devotion. Without them, this work would not have been possible. TABLE OF CONTENTS Page LIST OF TABLES --------------------------------------------------- vi LIST OF FIGURES -------------------------------------------------- vii LIST OF APPENDIX TABLES ------------------------------------------ viii LIST OF APPENDIX FIGURES ----------------------------------------- ix INTRODUCTION ----------------------------------------------------- 1 LITERATURE REVIEW ------------------------------------------------ 3 Staining with vital dyes as an index of viability ----------- 3 Macromolecular synthesis by the embryo ---------------------- 4 RNA synthesis ------------------------------------------ 4 Protein synthesis -------------------------------------- 6 Steroid metabolism by the embryo ---------------------------- 8 Energy metabolism of the embryo ----------------------------- 9 Oxygen consumption ------------------------------------- 9 Utilization of energy substrates ----------------------- 10 Carbon dioxide production ------------------------- lO Uptake of Z-deoxyglucose -------------------------- l2 MATERIALS AND METHODS -------------------------------------------- l3 Animals ----------------------------------------------------- 13 Production and collection of embryos ------------------------ l4 Staining with vital dyes ------------------------------------ 15 Evaluations of macromolecular synthesis --------------------- l5 Autoradiography --------------------------------------------- 16 Measurements of steroid uptake ------------------------------ l8 Metabolic assessments --------------------------------------- 18 Oxygen consumption ------------------------------------- 18 Carbon dioxide production ------------------------------ l9 2-Deoxyglucose uptake ---------------------------------- 20 Statistical analyses ---------------------------------------- 2T iv TABLE OF CONTENTS (continued) Page RESULTS ---------------------------------------------------------- 22 Staining with vital dyes as an index of viability ----------- 22 Macromolecular synthesis in the early embryo ---------------- 26 RNA synthesis ------------------------------------------ 31 Effect of ovulatory regimens ---------------------- 31 Protein synthesis -------------------------------------- 3T Steroid uptake of the early embryo -------------------------- 38 Effect of ovulatory regimens --------------------------- 38 Metabolism of the ovum and early embryo --------------------- 42 Oxygen consumption ------------------------------------- 42 Glucose utilization ------------------------------------ 42 Carbon dioxide production ------------------------- 42 2-Deoxyglucose uptake ----------------------------- 44 DISCUSSION ------------------------------------------------------- 47 SUMMARY AND CONCLUSIONS ------------------------------------------ 54 LITERATURE CITED ------------------------------------------------- 56 APPENDIX --------------------------------------------------------- 70 A. Biochemical evaluation of the unfertilized human oocyte 70 8. Publications by the author ----------------------------- 74 C. Vita --------------------------------------------------- 76 Table la lb LIST OF TABLES Validation of trypan blue (TpB) and fluorescein diace- tate (FDA) in hamster ova ------------------------------ Validation of trypan blue (TpB) and fluorescein diace- tate (FDA) in hamster ova .............................. Validation of vital dyes: In vitro development of hamster embryos from the one-to two-cell stage --------- Vital dye assays: Correlation with 3H-uridine incor- poration in Squirrel monkey ova ------------------------ Vital dye assays: Correlation with 3H-leucine incor- poration in squirrel monkey ova ------------------------ 3H-Uridine incorporation with respect to the time of oocyte collection in the squirrel monkey --------------- Incorporation and uptake of 3H-uridine by preimplanta- tion hamster embryos: Effect of superovulation -------- Uptake of 3H-estradiol T78 and 3H-progesterone by pre- implantation hamster embryos: Effect of superovulation Uptake of 3H-estradiol l7B and 3H-progesterone by un- fertilized and jn_vitro fertilized ova from squirrel monkeys: Effect of ovulation regimen ------------------ 2-Deoxyglucose uptake by squirrel monkey ova that are unfertilized, in vitro fertilized or degenerate -------- vi Page 23 24 27 30 32 34 37 4O 4T 46 Figure 4a 4b 7a 7b LIST OF FIGURES Page Statistical correlation between TpB and FDA ------------ 25 Effect of 3H-uridine concentration on incorporation and uptake by embryos ---------------------------------- 28 3H-Uridine incorporation in two-cell hamster embryos with respect to various treatments --------------------- 29 Autoradiograph of unfertilized control oocytes from a squirrel monkey ---------------------------------------- 33 Autoradiograph of unfertilized monkey oocytes which were suboptimally frozen ------------------------------- 33 3H-Uridine incorporation by early embryos of squirrel monkeys fertilized in_vitro ---------------------------- 35 3H-Leucine incorporation by early embryos of squirrel monkeys fertilized jn_vitro ---------------------------- 36 Autoradiograph of a two-cell hamster embryo which was sectioned and incubated for 2 hr in medium supple- mented with H-estradiol I78 --------------------------- 39 Autoradiograph of a two-cell hamster embryo which was sectioned and incubated for 2 hr in medium supple- mented with 3H-estradiol T78 and washed for l.5 hr in a lOOO-fold excess of nonradioactive estradiol-l78 ------- 39 Steroid uptake by early embryos of squirrel monkeys fertilized jn_vitro ------------------------------------ 43 2-Deoxyglucose uptake by unfertilized oocytes from squirrel monkeys: Effect of insulin ------------------- 45 vii LIST OF APPENDIX TABLES Table Page l Morphologic data on human oocyte recoveries ------------ 67 2 3H-Uridine incorporation and viability with respect to the time of oocyte collection in humans ---------------- 68 viii LIST OF APPENDIX FIGURES Figure Page l Photograph of a human oocyte recovered l2 hr after HCG administration ------------------------------------- 69 ix INTRODUCTION Biochemical analyses of embryos have been previously used to test viability in nonprimate systems or to investigate and establish normal metabolic pathways. However, little research has focused on the bio- chemistry of preimplantation development of primate embryos, particu- larly those embryos derived from ifl_yjtrg_fertilization. The present studies were designed to allow assessment of the embryo's metabolic requirements in an effort to maximize output of jn_yitrg fertilization systems utilizing primates. The embryos of subprimate species experience little difficulty in developing to the blastocyst stage in_yjtrg, Achieving this in nonhuman primates is more difficult. The aims of the present studies, then, were to determine both the viability and metabolism of primate embryos. Several biochemical estimates of such variables were used: (l) Assessment of viability of squirrel monkey embryos through the use of vital dyes; (2) Evaluation of 3H-uridine and 3H-leucine incorporation in early preimplantation development; (3) Determination of uptake of steroid hormones by squirrel monkey embryos through early preimplantation development; and 2 (4) Ascertainment of the level of utilization of metabolic substrates by monitoring oxygen consumption, carbon dioxide production from glucose and 2-deoxyglucose uptake by primate 0V6. LITERATURE REVIEW Staining with vital dyes as an index of viability Various vital dyes have been utilized to determine the viability of ova and other cells. These included acridine orange, a fluorescent dye (Austin and Bishop, 1959; Ezzell and Szego, 1979), several cyto- plasmic and nuclear stains (Dolan, 1965; Whittingham, 1978), trypan blue (Tennant, 1964; Clines §t_al,, 1980) and fluorescein diacetate (Rotman and Papermaster, 1966; McGrath gt_al,, 1975; Jarnagin and Luchsinger, 1980). Trypan blue (TpB) has proven a successful indicator of membrane integrity of cumulus and granulosa cells and embryos (Campbell, 1979; Peluso et_al:, 1982; Thadani gt__l,, 1982). Campbell (1979) demon- strated a 1:1 correlation between TpB exclusion and autoradiographic assessments of relative syntheses of DNA, RNA and protein by granulosa cells. Degeneration or atresia of ovarian follicles has also been characterized through the use of a viability index for granulosa cells with TpB (Peluso gt_al,, 1982). The best index of embryo viability currently available delineates the embryo's ability to take up diactyl fluorescein (FDA), a non- fluorescent compound (Church and Raines, 1980; Renard et_gl,, 1982; Taylor, 1982). Theoretically, once inside the cell, non-specific esterases cleave the acetate groups from FDA, converting it to the fluorescent compound, fluorescein, which is polar. Bright green 4 fluorescence under high-energy blue or ultraviolet light has been highly correlated with the ability of oocytes to mature in culture and embryos to go to term after transfer to recipients (Mohr and Trounson, 1980; Peluso gt a1 , 1982). Macromolecular synthesis by the embryo Measurements of synthesis of macromolecules (e.g., RNA and protein) have provided indications of overall metabolic competency of the ovum and embryo. I vivo uptake of 3H-uridine was demonstrable in ovarian u—-—- oocytes of rats, Macaca mulatta and M, fascicularis (Baker et_al,, 1969). RNA was synthesized at a high level by growing mouse oocytes (Bachvarova, 1981). This synthesis was still present, but reduced, in oocytes taken from antral follicles prior to and during germinal vesicle breakdown in mice, monkeys, cattle and swine (existing pri- marily as HnRNA, rRNA and tRNA) (Oakberg, 1968; Baker §t_al,, 1969; Bloom and Mukherjee, 1972; Bachvarova, 1974; Rodman and Bachvarova, 1976; Wassarman and Letourneau, 1976; Wolgemuth-Jarashow and Jagiello, 1979). Following fertilization, chromatographic and electrophoretic analyses have detected RNA synthesis by the one-cell stage in the mouse (as poly(A)' RNA (HnRNA precursor), poly(A)+ RNA (mRNA) and tRNA) and at the two-cell stage (as HnRNA of high molecular weight (mRNA precursor), rRNA, some mRNA and 4sRNA (Woodland and Graham, 1969; Murdoch and Wales, 1971; Knowland and Graham, 1972; Epstein, 1975; Clegg and Pik6, 1982). All classes of RNA were synthesized from the 8-cell stage through blastocyst (including mRNA (Warner and Hearn, 1977b). Prominent rRNA peaks were discovered at the 8-16 cell stage 5 (day 2 of embryonic development, when true nucleoli were labelled with 3H-uridine (Mintz, 1964)). There was a further increase in RNA syn- thesis from morula to blastocyst stage (day 3) (Ellem and Gwatkin, 1968; Pik6, 1970; Tasca and Hillman, 1970; Epstein and Daentl, 1971; Warner and Hearn, 1977a). 3 H-Uridine incorporation which was actino- mycin D-sensitive increased 90-fold at mouse morula and blastocyst stages compared to unfertilized controls (Monesi and Salfi, 1967). At low actinomycin 0 concentrations, RNA polymerase I and therefore rRNA synthesis of mouse embryos were preferentially blocked. There was no effect on development until concentrations of actinomycin D exceeded 0.1 ug/ml (Thomson and Biggers, 1966; Tasca and Hillman, 1970). This led some workers to conclude that little concurrent synthesis of RNA was necessary for protein synthesis in early cleavages of mouse embryos (Schultz, 1975; Brower and Schultz, 1982). However, much maternal message is lost as early as the two-cell stage in the mouse (Pik6 and Clegg, 1982) and dg_ngy9_synthesis of RNA may assume an important role thereafter. RNA synthesis (particularly mRNA and tRNA) in the rabbit embryo has not been demonstrated prior to the 16-ce11 stage (Schultz, 1973; Manes, 1977). Synthesis of RNA markedly increased by late cleavage stages (64-128 cells) (Manes, 1969, 1971). The RNA was present pri- marily as nucleolar rRNA (Manes, 1977). Quantitative assessments of total RNA and DNA content of oocytes and embryos have been determined by spectrophotometry (Olds g__al,, 1973; Henriet gt_al,, 1980). Innovative studies utilizing gene injection (plasmids produced by fusion of bacterial DNA and the regulator region from embryonic genes 6 of mice) have recently allowed us to further our understanding of regulation of transcription of the embryonic genome (Brinster gt_al,, 1982a; Palmiter et_al,, 1982a; Stewart e§_al,, 1982) and subsequent gene expression by progeny (Palmiter gt_al,, 1982b; Brinster et_al:, 1982b). Protein was found to be synthesized in oocytes from mouse and monkey follicles at the antral stage and undergoing meotic maturation, though synthesis was attenuated considerably (Baker et_als, 1969; Schultz et_al,, 1978, 1979). Much of the protein synthesis detectable at the two-cell stage in mouse embryos represents a control at the post-transcriptional level, utilizing mRNA's synthesized prior to fertilization (Braude gt al,, 1979; Cascio and Wassarman, 1982). There is little indication of an enhanced rate of protein synthesis at fertilization (Monesi and Salfi, 1967; Brinster, 1971a; Brinster et_al,, 1976; Abreu and Brinster, 1978). Only methionine incorporation (Schultz et_al,, 1979) and syn- thesis of the FPl-6 proteins were augmented (Cascio and Wassarman, 1982). In fact, there is some evidence for decreased protein synthe- sis with fertilization (Chen gt_al,, 1980). Qualitative patterns remain essentially unchanged in early embryo development (Van Blerkom and Brockway, 1975; Schultz gt 21,, 1979). However, utilizing a double-isotope labelling technique (3H- and 35 S-methionine) and two- dimensional polyacrylamide gel electrophoresis (2-D PAGE), Chen et_al, (1980) have compared 95 individual proteins prior to and subsequent to fertilization. These workers demonstrated significant increases in synthesis of only 6 specific proteins and a decline in the rates of 11 7 proteins. Amino acid incorporation increased as embryonic development proceeds further in the mouse (Mintz, 1964; Monesi and Salfi, 1967; Tasca and Hillman, 1970; Brinster, 1971a) and pig (Motlik g__al,, 1980). Amino acid incorporation (Epstein and Smith, 1973; Brinster gt 35S-methionine (which was Na+-dependent and 21,, 1976) and uptake at competitive) (Kaye gt_al,, 1982) increased several-fold between day 2 (two-cell) and day 4 (blastocyst). Autoradiographic techniques have been used to demonstrate 3 H-lysine incorporation at syngamy in porcine embryos with nuclear label disappearing by the four-cell stage (Motlik et_al,, 1980). Nuclear methionine and tryptophan were still present at 4- and 8-ce11 stages, respectively. Tryptophan was presumably incorporated into non-histone proteins assuming a role in genomic regulation. Studies of amino acid incorporation must consider the pool size of the endogenous precursor, which increases concomitantly with em- bryonic development (Sellens _t_;l., 1981; Schultz et al,, 1981). Therefore, uptake values are of major importance. With long culture periods (>13 hr), protein degradation also significantly increased by the blastocyst stage (Merz et_al,, 1981). Several specific proteins present in embryos have been analyzed. Tubulin synthesis in mouse embryos rose slightly after fertilization, increased to l4-fold by blastocyst stage and accounted for 2% of the total protein synthesis (Abreu and Brinster, 1978; Schultz gt al,, 1979). Actin, synthesized by the unfertilized mouse oocyte (Osborn and Moor, 1982), increased 10-fold by the 8-cell stage and 90-fold by the blastocyst stage. This represented 5.7% of the total protein synthesis. High levels of 8 alpha-fetoprotein, transferrin, fetuin and glycoprotein synthesis have 35 been measured by radioimmunoassay, S-methionine and 3H-leucine incorporation and 2—D PAGE before and after implantation in mouse, bovine, sheep, and pig embryos (Janzen et_ 1., 1982; Godkin gt al., 1982; Masters gt_al., 1982; Adamson, 1982). ——- Steroid metabolism by the embcyg Embryos of several species lack the capacity for steroid synthe- sis and metabolite interconversions until the peri-implantation period (Dickmann and Dey, 1974; Dickmann, 1979; Gadsby et_al,, 1981). Acetate incorporation into cholesterol by mouse embryos was enhanced ig_vitr9_from the blastocyst to the early somite stage (Carson gt_al,, 1982). These embryos were capable of converting 3H-pregnenolone to progesterone and acylpregnenolone. Rabbit blastocysts showed high aromatase activity by day 6 of development (Hoversland §t_al,, 1981; Wu and Lin, 1982b). Other lipids were also synthesized. Enhanced fatty acid levels were present by days 11-13 in bovine blastocysts (Menezo gt_al,, 1982). PGE2 and PGan were produced from 3H-arachi- donate by days 14-19 (Lewis gt_al,, 1982). Although many workers have demonstrated steroid synthesis by the embryo, only a few have attempted to assess radiosteroid uptake by the embryo (Smith, 1968; Bhatt and Bullock, 1974; Wu and Lin, 1982a). These studies were not validated autoradiographically. It is known that an appropriate hormonal milieu is required for proper development of embryos (Stone gt_al,, 1977; Dickmann gt al., 1977; Warner and Tollefson, 1978), and early embryos initially respond to endogenous levels of maternal steroids (Smith, 1968; Weitlauf and Greenwald, 9 1968; Smith and Smith, 1971). Responses to these steroids may be measured in terms of RNA synthesis, since steroid hormones classically cause derepression of genes (O'Malley gt _l,, 1973). However, workers have failed to demonstrate increases in incorporation of 3H-uridine by mouse embryos treated with estrogen or progesterone, in vitrg_(Warner and Tollefson, 1977, 1978). Estrogen has only been shown to increase uptake of amino acids by implanting blastocysts (Smith and Smith, 1971). Therefore, these studies suggest changes of a mitotic or membrane nature being effected. Energy metabolism of the embryo Oxidative pathways involved in substrate metabolism (primarily glucose) have been elucidated for ova from various species. Oxygen consumption increased during gonadotropin-induced maturation of un- fertilized rabbit and rat oocytes (Lindner gt_al,, 1974; Dekel gt 31., 1976; Magnusson et_al,, 1977; Magnusson and Hillensjb, 1981; Magnusson gt__l,, 1981). Fertilized ova from rabbits showed increased oxygen utilization during preimplantation development (Fridhandler, 1961). Consumption of oxygen by rabbit embryos reached highest levels at the blastocyst stage, primarily supporting the Na+-K+-ATPase used in active transport of ions (Benos and Balaban, 1980). Mills and Brin- ster (1967) and Sugawara and Umezu (1961) demonstrated a 3.5-fold increase in oxidation by the mouse blastocyst over that of the unfer- tilized egg. Further significant increases occurred at the 8-cell and subsequent stages. Oxygen consumption has been assessed simultaneously with CO2 pro- duction (see below; Brinster, 1968; Hammerstedt, 1975). The resulting 10 respiratory quotient was an important indicator of overall metabolic competency and preference for energy substrate. Measurements of 14 CO2 production from universally- or Specifi- cally-labelled glucose have been used to determine the metabolic pathways utilized by the preimplantation embryo (Brinster, 1967a). Carbon dioxide production increased lOO-fold over the first five days of development in the mouse, with a five-fold increase occurring at the time of fertilization. Incubation with specifically-labelled glucose (14C in the C-1 or C-6 position) resulted in a C-l/C-6 ratio of 1.6, indicating an active TCA cycle early in mouse development (Brinster, 1967a). The Embden-Meyerhof pathway does not assume a prominent role until the last two days before implantation (Thomson, 1967). Conversely, in the rabbit, inhibitors of oxidative metabolism have demonstrated both an active pentose shunt (Cl/C6 = 10 one day after fertilization, then declines) (Fridhandler, 1961; Wales and Whittingham, 1970; Wales, 1973) and an active TCA cycle in preimplan- tation embryos (Quinn and Wales, 1973a; Kane and Buckley, 1977). Glycolysis was not quantitatively observed until the blastocyst stage (Wales and Whittingham, 1970). Experiments comparing oxidation of various substrates proved pyruvate to be the primary energy substrate utilized by two-cell mouse embryos (Biggers gt_gl,, 1967) and primate oocytes (Brinster, 1971b). Brinster (1971b) demonstrated a greater capability for pyruvate oxidation for the monkey oocyte than for the mouse ovum. LDH activity was, however, 80-fold greater in mouse oocytes than either rabbit or primate (rhesus monkey, squirrel monkey and human) oocytes (Brinster, 1967b). The values for pyruvate and glucose oxidation by primate oocytes were equivalent to those of the 11 unfertilized rabbit ovum (Brinster, 1968, 1969), but two- to five-fold greater than for the unfertilized mouse ovum (Brinster, 1967a). This may have been attributable merely to oocyte size, as the volume of rabbit and primate ova is roughly 3.5 times that of the mouse (Brin- ster, 1971b). Metabolic pathways have also been elucidated through the use of inhibitors of carbohydrate, protein and nucleic acid meta- bolism (Thomson and Biggers, 1966; Thomson, 1967). Comparisons of viability between mouse blastocysts developing in yjt:9_and ig_yiy9_have been made by monitoring C02 production (Menke and McLaren, 1970). The mean C02 output and trophoblast outgrowth of blastocysts (highly correlated) cultured jn_yitrg_from the 8-cell stage were significantly reduced when compared to jn_yjy9_controls. The addition of fetal calf serum to the medium augmented both viabi- lity indices. Other metabolic determinations of embryo viability have included ATP content, and pyruvate and lactate uptake (Quinn and Wales, 1973b). A11 indices were diminished with retarded embryonic development. In yitrg_uptake by bovine blastocysts has proven to be a good index of subsequent development jn_yjyg_(Renard gt 21,, 1980; Renard gt_al,, 1982). Of 13 transferred embryos which took up more than 2.5 pg glucose/embryo/hr, 69% maintained pregnancy at day 50. Only 14% of ova considered non-viable by the assay did so. 3H-Glucose uptake increased over days 5-7 post-coitus in the rabbit blastocyst (Benos and Biggers, 1981). This uptake was not dependent on external sodium. Glucose utilization, and hence, the cellular metabolic rate of various mammalian tissues have been assessed by the cells' ability to 12 take up radiolabelled 2-deoxy-D-glucose (2—DG) (Sokoloff gt_al,, 1977; Van den Broeck and Van Steveninck, 1981; Astic and Saucier, 1982). 2-DG is a nonmetabolizable analogue of glucose that, depending on circumstances, is transferred across cell membranes by facilitated transport (Kotyk and Michaljanicova, 1974) (like fructose, mannose and glucose outside kidney and intestinal epithelium (White, Handler and Smith, 1968)) or, as is more often the case, by active transport (Jasper and Van Steveninck, 1975) (utilizing the same carrier as galactose (Parra gt_al,, 1980) and 3-O-methylglucose (Segal and Ingbar, 1980)). 2-DG is phosphorylated by glucokinase (like mannose and glucose) to 2-deoxyglucose-6-phosphate (2-DG-6P). 2-DG-6P inhibits glucose phosphate isomerase and cannot be further metabolized. Analy- ses of 2-DG uptake therefore provide valid information about the utilization of glucose (Brooks, 1982). METHODS AND MATERIALS Animals Forty mature female hamsters (Mesocricetus auratus) (8-10 weeks of age) were monitored for postovulatory vaginal discharge for at least three cycles before being placed on experiment. Hamsters nor- mally received a superovulatory regimen of 30 I.U. pregnant mare's serum (PMS) (Serotropin, Teizo, Tokyo, Japan) i.p. on the morning of Day 1 (day of ovulatory plug). This was followed by 30 I.U. HCG (A.P.L., Ayerst Laboratories, Inc., NY) i.p. 76 hr later (Mizoguchi and Dukelow, 1980). In the experiments on steroid uptake, animals of group A received no exogenous gonadotropins, and ovulated naturally. All animals, regardless of treatment, were then mated on the evening of Day 4. Following mating, animals were sacrificed at varying times to obtain embryos at different stages of development. Squirrel monkeys (Saimiri sciureus) of Bolivian and Guyanan origin (Primate Imports, Charles River, Inc., Port Washington, NY) were housed and fed as previously described (Kuehl and Dukelow, 1979). Adult females of the same subspecies were used for related studies (Bolivian type for macromolecular and steroid studies, Guyanan type for metabolic studies). Animals were exposed to fluorescent lighting on a 14L:100 cycle with ambient temperature controlled at 2112°C. In the present experiments, animals were used during the breeding season (October through May). They normally received an ovulatory regimen of 13 14 4 days of 1 mg of FSH i.m. daily (Burns-Biotec Laboratories, Inc., Omaha, NE). However, in group B in the experiments on steroid uptake, animals received 5 days of FSH. This reflected the hormonal regimen designed for induction of single and double ovulations in Saimiri (Dukelow, 1970), which requires increased FSH during the anovoulatory season (Kuehl and Dukelow, 1975). On the final day of FSH treatment, 250 I.U. HCG was administered i.m., 16 hr prior to laparoscopy for follicular aspiration (Dukelow, 1979). Production and collection of embryos Hamster embryos were recovered at 40 hr after mating for two-cell embryos and 60 hr for four-cell embryos from the oviduct and at 72 hr for eight-cell embryos, 78 hr for morulae and 86 hr for blastocysts ‘from the uterus (Ghosh e__al,, 1982). Squirrel monkey oocytes were recovered at laparoscopy (Kuehl and Dukelow, 1975, 1979). If processed immediately, oocytes were mechani- cally denuded of cumulus cells with glass pipettes (unless stated otherwise). Some oocytes were allowed to mature for 21 hr in tissue culture medium (TC-199, GIBCO, Grand Island, NY) which was modified to contain 1 mM pyruvate, 100 ug/ml gentamycin and 1 U/ml heparin (Asa- kawa gt 21,, 1982). Twenty percent fetal calf serum (FCS) (heat inactivated for 30 min at 56°C) was added prior to insemination jg_ yitrg, Semen was collected by electroejaculation (Kuehl and Dukelow, 1974). Embryos were collected 24 hr after insemination for one-cell, 48 hr for two-cell and 52-60 hr for three- to four-cell embryos. Prior to experimental processing, ova and embryos from one animal or group of animals were divided among the various treatments. 15 Techniques were usually validated with hamster ova (where larger numbers of embryos were available), and further modified for squirrel monkey ova. Some ova were also subjected to suboptimal (>0.5°C/min freezing and 500°C/min thawing rates, Leibo, 1977) and optimal (O.25°C/ min freezing and 3°C/min thawing rates, DeMayo §t_al,, 1983) freezing procedures to artificially yield groups of ova in varying states of degeneration. Staining with vital dyes Ova were transferred to depression slides containing 10 pl of 15 uM FDA in Dulbecco's phosphate-buffered saline (PBS) and held at room temperature for one min. The ova were then examined for 10 sec under a fluorescence microscope (Leitz 8612 and 8038 exciter filters and a K510 long pass barrier filter, The Microscope Co., New Castle, PA). Ova were classified as either positive (bright fluorescence) or nega- tive (faint or no fluorescence). Examinations were also made for exclusion or uptake by ova of 0.2% TpB. Ova were washed and further processed to correlate with morphologic studies and macromolecular synthesis. Evaluations of macromolecular synthesis Ova and embryos were incubated in 0.25 ml of 2.8 (Daentl and Epstein, 1971) and 5.6 uM 5-3H-uridine (for hamster and squirrel monkey ova, respectively; S.A. 18 Ci/mmole) which supplemented the modified TC-l99. Incubations took place for 3 hr in an atmosphere of 5% CO2 in air. Ova and embryos were washed 10X in medium containing unlabelled uridine in lOOO-fold excess and solubilized in 100 pl of 16 0.14 M 2-mercaptoethanol and 0.1% sodium dodecylsulphate in phosphate buffer (pH 7.4) (Fishel and Surani, 1978). Samples were heated to 65°C for one hr in a water bath, duplicate aliquots removed and added to Whatman GF/C glass fiber filters (Fisher Scientific, Pittsburgh, PA), and air-dried. RNA was precipitated by addition of 30 m1 cold 10% TCA and 30 m1 cold ethanol (EtOH) to the discs under light suction filtration (Sartorius-Membranfilter, Gdttingen, W. Germany). Treat- ments of selected filters with either ribonuclease (RNase) A from bovine pancreas (0.4%, Sigma, St. Louis, MO) and 0.5 N NaOH reduced 3H-uridine incorporation into TCA-precipitable material to background levels, thereby serving as controls. Total uptake was determined by assaying discs without prior TCA/EtOH treatment. Discs were assayed for radioactivity in 10 m1 ACS (Amersham, Arlington Hts., IL) in a Searle Model 6891 liquid scintillation counter. Machine efficiency was 64.5% for 3H and quenching was 5.7% with an external standard. Aliquots of the final wash were processed as procedural blanks (usu- ally 30-60 CPM). The moles of precursor incorporated/embryo/unit time were calculated using the known specific activity of the precursor and the incorporated radioactivity (Epstein, 1975). Autoradiography Squirrel monkey ova were incubated 3 hr in modified TC-199 sup- plemented with 5.6 uM 5-3H-uridine (S.A. 18 Ci/mmole) or 0.4 uM L- 4,5-3H-leucine (S.A. 50.4 Ci/mmole) and treated as described above. Following washing, ova were fixed 24 hr in Bouin's fluid and processed for autoradiography (AR) (Weitlauf and Greenwald, 1971). Ova were 17 embedded in paraffin and serially sectioned at 5 pm. For uridine, alternate paraffin sections were mounted on two sets of slides and the paraffin removed. One set was treated with RNase A (0.1 mg%, activity 85 Kunitz units/mg, Sigma, St. Louis, MO) in sodium phosphate buffer (0.1%, pH 7.4). The other set of slides received only buffer treat- ment. A11 slices were incubated at 37°C for 1 hr and were subse- quently treated with 5% TCA for 10 min at 4°C. For leucine, since Bouin's fixative removes unincorporated amino acids (Weitlauf and Greenwald, 1971), no further treatment was required. All slides were washed 15 min in tap water, air-dried and dipped in Kodak NTB-3 emul- sion at 42°C in the dark. The slides were exposed two weeks, de- ve10ped, fixed and stained with hematoxylin and eosin (Peluso and Hutz, 1980). Two blank slides in each set were exposed to light and two processed in the dark to control for negative and positive chemo- graphy, respectively. 3H-Uridine and -1eucine incorporation into RNA and protein, respectively, were determined by counting reduced silver grains over three different areas of 300 um2 each of nucleoplasm or cytoplasm with a micrometer reticle. These counts were averaged and background counts from similar averaging of equivalent areas 200 um from the ovum were subtracted. (For the case of uridine, the back- ground counts were the same as RNase-treatment.) The area over which grains were counted was converted to 1000 um2 for ease in calculations and graphic representation. Steroid autoradiography was according to Uriel gt a1, (1973). In brief, slides of sectioned ova were incubated 2 hr in PBS containing 18 3 0.005 ug/ml of either 2,4,6,7-3H-estradiol—l78 (E or 1,2,6,7- H- 2) progesterone (P) (S.A. 94 Ci/mmole each), washed 1.5 hr in running tap water and autoradiographed as described above. Positive controls were nonradioactive E2 and P (each in 1000-fold excess) to compete for radioactive E2 and P, respectively. Measurements of steroid uptake 3H-Estradiol or 3H-progesterone (S.A. 94 Ci/mmole each) were dried and dissolved in 0.1 ml EtOH. Ova were then incubated for 2 hr in 0.2 m1 of modified TC-199 supplemented with 0.06 uM of either radioactively-labelled steroid. (This amount provided for maximum uptake in preliminary trials). Ova were washed 10X in phosphate buffer, dissolved in 0.1 m1 of tissue solubilizer (Soluene, Packard, Downers Grove, IL), and the solution assayed for radioactivity as described above in 10 m1 of methanolic AC5 to reduce chemilumines- cence. Metabolic assessments Oxygen consumption Oxygen consumption of ova was measured by the method of Benos and Balaban (1980). This utilized a polarographic oxygen electrode of the Clark type with micromodifications (Model 5331, Yellow Springs Instru- ments, Yellow Springs, OH). Hamster ova or squirrel monkey oocytes with cumulus were incubated for 4 hr in a sealed glass chamber (0.5- 0.8 ml volume) containing modified TC-l99 medium (Seamark g__gl,, 1976). The medium, previously equilibrated with 5% CO2 in air, was surrounded by a water jacket controlled at 37:0.01°C by a Haake Model 19 FE2 circulating water bath (Haake, Karlsruhe, W. Germany). The chambers' contents were stirred continuously with magnetic stirrers and additions made with a microsyringe through the access ports. Oxygen consumption of ova was calculated from the observed decrease in 02 tension per unit time (YSl 02 Electrode References, 1974) as com- pared to a control chamber containing no ova. A positive control was the addition of 1x10"4 M KCN, which reduced the oxygen consumption of ova to baseline levels. Consumption of oxygen by squirrel monkey oocytes was estimated by taking 10% of the total uptake of the oocyte- cumulus complex as the approximate value for oocytes alone (Dekel gt §_l_.,1976). Carbon dioxide production CO2 production by oocytes was monitored by modifications of pub- lished techniques (Brinster, 1967; Menke and McLaren, 1970) to those used by Dey §t_al, (1979). Oocytes from squirrel monkeys were incu- bated for 4 hr at 37°C in 5% CO2 in air in Dulbecco's PBS (titrated to pH 7.3 with 0.5 N NaOH), supplemented with 100 pg/ml gentamycin. (PBS has been shown to lack embryotoxic or metabolic effects in a 4 hr culture period (Quinn and Wales, l973b,c).) The only energy sub- 4 4M D_U_14 strates added were 1.80x10' or 5.56x10' C-glucose (S.A. 296 mCi/mmole). Incubations were in equilibrated test tubes sealed with rubber stoppers, and containing plastic center wells (Kontes Glass- ware, Vineland, NJ). The incubations were terminated and 14CO2 liberated from the medium at the end of the culture period with an injection of 0.25 ml 0.05 M potassium hydrogen phthalate buffer (pH 4.0). NCS (0.25 ml, Amersham, Arlington Hts., IL) was then injected 20 14 into the center wells to absorb the C02. The tubes were agitated one hr in a metabolic shaker (100 cycles per min). The tubes were 14 then discarded and the center wells containing the CO -1abelled NCS 2 were dropped into liquid scintillation vials with 10 ml standard PPO- POPOP (6 g/L and 75 mg/L, respectively, in toluene; Spectrafluor, Fisher Scientific, Pittsburgh, PA). Efficiences and quenching were assessed using a NaZMCO3 standard. Picogram-atoms of carbon incor- porated were calculated utilizing the method of Menke and McLaren (1970). Blanks contained all components except oocytes. Background levels produced 100-200 cpm, significantly less than experimental tubes. 2-Deoxyglucose uptake Ova from squirrel monkeys were preincubated one hr in 0.2 ml TC- 199 containing 5.56 mM D-glucose (non-radioactive) and washed in saline 5 times. They were then incubated in 0.2 m1 of modified PBS containing 1 uM 2-deoxy-D-1-3H-glucose (S.A. 25 Ci/mmole) (Dunn and Mallucci, 1980) as the only energy substrate. (Incubations with 2- deoxyglucose had no detrimental effect on jg_yjtrg_development of early embryos (Kane and Buckley, 1977) or on viability of other cell types as assessed by trypan blue (Segal and Ingbar, 1980).) Ova were cultured for 3 hr with and without the addition of 10 nM or 1 UM insulin (Segal and Ingbar, 1980) from bovine pancreas (24.3 U/mg; ICN Pharmaceuticals, Cleveland, OH). Following incubations, ova were washed 10X in nonradioactive medium and solubilized in 100 pl of 0.14 M 2-mercaptoethanol and 0.1% sodium dodecylsulphate in sodium phos- phate buffer (0.1%, pH 7.4) for 1 hr at 65°C. Aliquots of the final 21 wash served as procedural blanks (Fishel and Surani, 1978). Machine efficiency for 3H was 57.6% and quenching was 5% using an external standard. Although 2-DG depresses ATP levels in some cell types, the technique allows accurate measurements of initial substrate utiliza- tion (Parra et_al,, 1980). Statistical analyses Since scintillation counts normally follow a Poisson distribution (Steel and Torrie, 1980), such data were usually transformed (Vi) prior to further analysis. Steroid data were transformed to log (X+1). Comparisons were analyzed by Student's t-test, or randomized one-way ANOV or factorial designs. Student-Newman-Keuls test was used to compare multiple groups. The rank sum (Mann-Whitney U) and Kruskal- Wallis tests were performed if data was still nonparametric following transformation. Vital dye data were compared by x2 (contingency tables) or Fisher exact test, or by simple linear correlation. Percentage data were evaluated following angular transformation (sin-1 vGZ). Simple linear regression analyses were done to validate oxygen consumption data. P<0.05 was considered to be significant. RESULTS Control ova obtained from hamsters took up FDA and fluoresced brightly, excluded TpB, and appeared morphologically normal after a 3 hr culture period (Table 1A). These ova also incorporated 3H-uridine to a significant extent (Table 18). A suboptimal freezing procedure reduced all indices of viability to zero. Although an optimal freez- ing procedure for hamster ova reduced viability, ova judged normal by vital stain criteria incorporated and took up 3H-uridine to the same extent as controls. Although TpB exclusion and FDA uptake were normally highly correlated with each other in ova of both species over various treatments (r = 0.99, Figure 1), there were discrepancies in groups with intermediate viability after culture (Table 1A). In this third group, there was a drop of viability after culture (as assessed morphologically) to TpB levels before and after culture. The percen- tage of ova assessed viable by FDA was lower than the other indices both prior and subsequent to culture. There was no effect of incuba- tion with radioprecursor on the viability of control ova over the 3 hr. The use of vital dyes had no effect on radiouridine uptake by hamster two-cell embryos, as estimated by radioautography. Control embryos (no stains, n=4) had grain counts of 10815 versus 95:9 grains/ 2 1000 um for embryos incubated 1 min in each of TpB and FDA and 22 23 .Amo.ovav xppcmowwwcmwm memwu pawcumcmaam coesoo m mcwxomp so; a cwguwz mgmnE:z one .Amo.ovav asosm Pocucoo m>wuumammc Eogm wcmsmmmmc xpgcmuwewcmmm k. .wLau—SU $0 L: m Lmukm n .u.< .mgzppsu wLommn n .U.m — . .mwm . .mem . .mem . .mmm . .whm . “mam me_NeeeL esAo see mme esfl_ mmv ome esAN mev _Ne 34A, may oee eesam owv _ee eeeAm Nev Nae Peeeeeo .mwe . .mwe .mme .mww .mww . .mwe meeeeeea son o 1A0 NV m shov o sflov o ,on o sAo NV m _eeeeeeesm .pmh .pme . .pmw . .pme . hmm. hawk Aoopv om_ Aoo_v m. Am mmv see Am mmv me_ Am may ep Aoo_v om_ _oee:eu Aev .o.< Aev .o.m Axe .u.< Rev .u.m Aev P.u.< “xv r.u.m pcmEpmmL» Peace: xppmowmoposacoz wxmpga o gunman: cw A <_ m4m .mpm>m~ uczosmxomn op vmoanmc mpczoo cwmch _NHN numm Ae.wev __ Ae.mwv Ne Ae.mmv N_ e_ __ee-eee eeNeFeeeeL Pm.QMN nflmm Aw.emv m Am.emv w Am.emv m mm eeeepeeeeeea «QL\ mae «DL\ me» - + + - Aev Aev Aev_2:3_ mxmua: o xmxcoz Pmecwacm cw cowpmcoagoocH mcmuwcsuzm suwz :owpm_mccou ”mxmmm< oxo quw> m m4mmp uczocmxomn op umozcms macaou cemeu .u.m.H cams mew mmapm> F I. mczuwooga _N+m on o on o on o e_ meeNeeeL _eeeeeeesm _~Hmmm Aoo_v e_ Aoo_v e_ Aooev e_ e_ _oeeeeu Aev Lev Aevpeeez NE: ooo_\m:wmco mxmpa: o xmxcoz chcwscm c_ cowumgoasoocH mcwosmbix w m4m

33 .Aommxv Emmpaoo mcu mo mcwcwmum map use mcwmcm Lm>me cw mmmmgowc UFumEmsu on» mpoz .mcwozmriz cue: umpcmsm—aazm Ezwuwe cw L; m Lo; umumnsocw mew: mouxuoo .:w~osw z—Pmewpamnzm mew: gums: mmuzuoo zmxcos umequme:: to :nmgmowumcou:< . ommx oceuzopuz ;p_z umpcmsmpgasm Ezwuwe cw s; m com umpmnsucw mew: mmpzuoo m .zmxcos Fmecwzcm m Eoge mmuzuoo Fogpcou umNWFTHmec: we snmsmowumsopz< .nw .ov acumen 34 TABLE 5 3H-Uridine Incorporation with Respect to the Time of Oocyte Collection in the Squirrel Monkey Treatment Incorporation1 Uptake1 15 hr after HCG 3.81:0.54 (8)2 68.41:8.93 (8) 36 hr after HCG 1.07:0.14 (4)* 3.28:1.00 (4)* 1Femtomoles 3H-uridine/oocyte/3 hr : S.E. 2Number of trials in parentheses; 1-5 oocytes per trial. * Significantly different from respective group at 16 hr (p<0.05). 35 GRAINS/ 100mm? UNF IVF TWO- TH REE-/ FOUR CELL CELL C E LI. STAGE Figure 5. 3H-Uridine incorporation by early embryos of squirrel monkeys, fertilized in vitro. Autoradiographic assessments were done on a per-cell bEETS. Number of ova at base of bars. Values are expressed as mean :_S.E. *Significantly different from previous cell stage (p<0.05). 36 4oo "e I at o o 9 3. mo , Z .. < 5:5.__: 8 :=:-:5:5 o 1:: 1o 7 3 IMMATURE ' IVF - MATURE c151? CELL STAGE \ Figure 6. 3H-Leucine incorporation by early embryos of squirrel monkeys, fertilized ig_vitro. Autoradiographic assessments were done on a per-cell basis. Number of ova at base of bars. Values are expressed as mean :_S.E. *Significantly different from previous cell stage (p<0.05). 37 .Amo.0vav m>o umumps>oizppmsapmc mo azogm w>wuowamog Ease “cocowwwu x—pcmowmwcmwmtk .Amo.ovav mmwum Fpmo mzom>mca Eogm ucmcmmmwu Appcmuwmwcmwmt .m.m.H L; m\oxgnsw\mcwvwc:iz mmpoeousmd m .Amumxooammpn ucm mmpzcozv m .AFPmUimv opim .A—FmUiqv op .A—quimv omio— “macaw—am; Lon cwassz .moxsaem mo Longs: Pouch w _ me.F-H_e.mm .om.-Hee.em me o.enfloo.m__ oe.nuoe.em om memseoemeem .me.nuoo.em .No.mfloe._F o_ sm_.-Hm_.em 5mm.qumo.e_ mm ee_=eez Ne.-Hom.me ee.QHom.e oe .em.-Hoe.o_ em.quxe.m om F_ee-eemem 5mm.-flom.m 5mm.qweo.m om ee.mfle_.e om.omde.m oe _Fee-e=oa .sme.ouom.m om.QHe_._ om o..qwom.e Npe.qmwe.e me _Pee-oze mxmpa: cowumcoacoocH mxmua: cowamcoacoocH cowum_:>osma:m P2 cowgmpz>o Fecapmz Fz mmmum Fpmu cowwmrz>ocma=m mo pumCCM ”moxcnsm Lomeo: cowpmuceraswmca ha oneneszizm mo mxmpa: use cowpmconcoucH o m;m

o umum—:>oixppwgzum: Co azocm w>wuomgmog soc» pcmsowmwn appzeowmwcmwmts V .Amo.ovav macaw Fpmo maow>msa soc» acmcmmmwv xpucmowmwcmwmk .mumUWPamc Log mozcnem o_im ”mommcucwcma cw mmumuwpamc mo Longsz 40 N .m.m.H Le N\e»eeEo\zau oee meee=_ Aev mm.woooe --- +5Amv __.Hoom --- oepzeez +Amv Ne-flmem_ Amy mmnuoewm is v emmflemm_ .Aev Neeueoem _Foo-eemem seAev me-H_em .ANV NF.HmNPN esflev me-Mwom_ .Amv epnwmmmm __eo-esed Rev om.Heee Amy mmuflem__ ..Aev FF-ueem_ Nfimv _ee-HmmFN Peoo-eze covumpz>ogmasm Possumz cowpmpz>ogmaam chzwmz omeem F_ou mcocmpmmmocm QNP Powumsomm cowgmpz>ocwa=m we powwow cowumucmpaawwca zn mcosmummmocaiz m m m4m<~ ”moxsnEm smumsmz use amp Pownmcummiz m we mxmpa: 41 .Amo.ovav :md ea mxaa a Eye: aapamgp aaacm a>euaaamaL Eage acacaeeea zepcaoeeecmemee .Amo.ovav aaagm aaNeeepcaeaa Ease acacaeeea xeacmoeeecmeme .mpaaepaag can a>a mum ”mamazpcacaa ce a>a ea LaaEzz N .m.m.H L; N\E=>a\zao some men muecae .Aev Nweweem_ .Amv ma-Hmme_ “av emmflammm ARV maeuoomm Feoo-oeo eoNePeesod Ae_v FN-Hmee Aev am.flaae eeAF_V _eommmem Nae—v _Na-Hmm_F eoNepeeeoeea Ema amen m Ema amen e Ema oxen m Ind meme e omeem _eoa acacaamamaca nee Faeaaspmm cwmwmam caeuaez>o ea uaaeem "mxaxcaz Faeceaam Eace a>o aaNeeepLad acae> EH aca aaNe_eaLae:: ma acacaumamasaiz eee ee_ FoeeeLEma-z eo demon: m m w m4m. a a 5. \ 0.6 m m .1 C) 12 & CLZ 43 l j s8 \ \ $8885 \\\g§\\ L:\\:- ;\\.\\‘\\:S \\\\ , 3:» SQ, \ \ \:\.\ \‘ \ \ \\\\;\ \\\\\ \\\\ \\ \\\>‘ ‘ x\\ \\ // a ‘\§§.. n» ,\\\ \\ \\ ESTRADIOI. PROGESTERONE Steroid uptake by early embryos of squirrel monkeys, Number of ova at base of bars. Values are *Significantly different from previous cell stage (p<0.05). 44 trials, 9-10 oocytes per trial) and 4l.9:2.8 picogram (pg)-atoms glucose carbon/oocyte/4 hr (n = 4 trials), respectively. From the experiments on oxygen consumption and 14 14 CO2 production, an estimated molar ratio of 002 produced from universally-labelled glucose to total oxygen consumption was calculated at 0.19 for the unfertilized squirrel monkey oocyte. The addition of insulin at concentrations of 10 nM and 1 uM in- creased 2-deoxyglucose uptake by unfertilized oocytes from squirrel monkeys over controls, but not to a significant extent (from 13.95: 2.40 to 15.64:3.78 to 18.84:].22 femtomoles (fmoles)/oocyte/3 h, respectively) (Figure 9). There was no change of 2-DG uptake at fertilization (Table 9). All ova used in the 2-DG experiments were viable by TpB and FDA prior to and subsequent to the culture period. The uptake of 2-deoxyglucose by fertilized and unfertilized ova classified as degenerate by morphology and vital dyes was reduced to background levels (Table 9). 45 2()C> lCDCD PERCENT OF CONTROL comam lOnM lyM INSULIN Figure 9. 2-Deoxyglucose uptake by unfertilized oocytes from squirrel monkeys: Effect of insulin. Four to five trials were run per group, with 4-10 oocytes per trial. Values are expressed as mean + S.E. 46 TABLE 9 2—Deoxyg1ucose Uptake by Squirrel Monkey Ova that are Unfertilized, lg_Vitro Fertilized or Degenerate Percent Viable by 1 Cell Stage Vital Dyes 2-DG Uptake Unfertilized 100 (5)2 14.531120 Fertilized one-cell 100 (2) 16.67:2.60 Degenerate O (3) ---3 1Femtomoles 2-DG/ovum/3 hr :_S.E. 2Number of trials in parentheses; 3-10 ova per trial. 3Scintillation counts reduced to background levels. DISCUSSION The vital dyes trypan blue (TpB) and fluorescein diacetate (FDA) were validated for use with hamster and primate ova in the present studies. Exclusion of TpB and positive fluorscence with FDA by ova in various treatments correlated well with 3H-uridine and -1eucine incor- poration and stability and development jg ngrg, Such correlations have also been shown for embryos and granulosa cells of other mammals (Campbell, 1979; Mohr and Trounson, 1980; Peluso g:_gl,, 1982). Over a sufficiently long period of culture, particularly in groups with intermediate viability, FDA appears a better predictor of viability than either TpB or purely visual assessments of morphology. However, as there exists such a high statistical correlation between TpB and FDA, it would be unnecessary to utilize both together in the future. Although FDA may be a more active indicator of viability, the assay also requires expensive fluorescence equipment and is more time- consuming than TpB. Therefore, TpB should suffice for most quick assessments of embryo viability jg_yj:rg, Nevertheless, both vital dyes are excellent indicators of viability of primate embryos produced by in yi:rg_fertilization. The decrease in 3H-uridine incorporation by oocytes at 36 hr after HCG administration to squirrel monkeys is consistent with the 47 48 decrease seen with intrafollicular maturation in mammalian oocytes (Baker g:_gl,, 1969; Bachvarova, 1974; Wassarman and Letourneau, 1976). Autoradiographic analysis showed an increase in the capacity of squirrel monkey ova to incorporate 3H-uridine after jg_yi:rg_ferti- lization. There was a further increase again after the second cleav- age division. However, the amount of 3H-uridine incorporated was several-fold lower than in similar autoradiographs of two-cell hamster embryos. This difference may be due to appreciable differences in precursor pools or membrane permeabilities to precursors (Clegg and Pik6, 1982). Nevertheless, relative RNA synthesis remains quite low in the early primate embryo. This observation compares with the low synthesis of RNA detectable at early stages in the mouse (Knowland and Graham, 1972). In this species, major increases in RNA synthesis do not occur until the 8-cell and morula stages, with further increases at the blastocyst stage (Monesi and Salfi, 1967; Ellem and Gwatkin, 1968). The decreases in 3H-leucine incorporation with oocyte matura- tion and the low levels at fertilization and first cleavage are quali- tatively similar to that demonstrated for other mammalian species (Brinster, 1971a; Schultz g£_gl,, 1978, 1979; Chen g§_al,, 1980). These attenuated levels of relative protein synthesis correlate with the observed low number of polyribosomes up to the morula and blasto- cyst stages of primate embryos fertilized jg_yiyg_(Enders and Schlafke, 1981) and ig_yi:rg (Yorozu e:_gl,, 1983). Again, as the size of the precursor pool for leucine is not known in primate ova, absolute measurements could not be made. Apparent increases in protein synthe- sis occur late in preimplantation development, primarily at 8-ce11 and 49 blastocyst stages in the mouse (Epstein and Smith, 1973; Abreu and Brinster, 1978; Schultz 22.91:, 1979; Kaye e:_al,, 1982) and at the blastocyst stage and beyond in embryos of domestic animals (Godkin 33 al,, 1982; Janzen §:_al,, 1982). RNA synthesis is augmented throughout preimplantation development in mammalian embryos (Epstein, 1975; Clegg and Pik6, 1982). Our values for the relative incorporation of 3H-uridine in hamster embryos closely resemble those for the mouse (Daentl and Epstein, 1971). (Absolute values for 3H-uridine incorporation (and RNA synthesis) cannot be given, as the size of the embryonic UTP pool is unknown for any mammalian species but the mouse (Clegg and Pik6, 1977).) However, few studies have attempted to assess steroid uptake by embryos (Smith, 1968; Bhatt and Bullock, 1974; Wu and Lin, 1982a). The present studies demonstrate definite uptake of E2 and P by ova from hamsters and squirrel monkeys and changes with in_yj:rg_development. The classical mode of steroid action is by gene derepression and activa- tion of RNA synthesis (O'Malley g: 31,, 1973). One can therefore expect changes in 3H-uridine incorporation through the use of various ovulatory regimens as compared to normal ovulatory cycles. This hypothesis was based on the observation that treatment with exogenous gonadotropins alters levels of endogenous steroids in several species (Greenwald, 1976; Schrams g: 21,, 1979; Edwards g:_gl,, 1980). In the present study, varying the length of FSH treatment showed no appre- ciable effects on steroid uptake in embryos from squirrel monkeys. However, the superovulatory regimen given hamsters significantly decreased uptake of exogenous radiosteroids by the embryos at 50 virtually all stages analyzed. Yet, there was no concomitant effect on relative incorporation of 3H-uridine. Absence of effects of exo- genously administered steroids on RNA synthesis of mouse embryos, jg_ ngrg, was demonstrated by Warner and Tollefson (1977, 1978). These investigators hypothesized that the effects of E2 and P are directly or indirectly on the membrane to alter permeability and subsequent embryonic cleavage. This hypothesis is yet to be confirmed. Changes in embryonic steroid uptake and receptors may therefore not be medi- ated by alterations in RNA synthesis. The apparent reduction in steroid uptake by superovulated hamster embryos may be attributed to saturation of embryonic receptors with augmented levels of endogenous steroids. (Steroid uptake, however, is not necessarily indicative of receptor number (Martel and Psychoyos, 1981; Logeat g:_al,, 1982).) Such saturation of receptors would alter true uptake values of radiosteroids. This has been reported to occur in studies of steroid uptake at implantation sites in pregnant mice and rats (Sartor, 1977; Ward g:_al,, 1978). (In preliminary studies in our laboratory, we administered 3H-E2 and 3H-P to pregnant hamsters carrying embryos at varying stages of preimplantation development and demonstrated steroid uptake by the reproductive tract and embryos. Relative changes between the jg_yi:rg_and jg_yjyg studies were identi- cal, suggesting ig_vivo saturation of steroid receptors.) An alterna- tive hypothesis is an actual alteration in receptor sites such as occurs in down-regulation in the presence of prolonged, elevated levels of endogenous hormones (Savoy-Moore g:_gl,, 1980). Yet another 51 hypothesis is a delay in receptor synthesis which was not detected as a concomitant change in RNA synthesis at the level investigated. Although changes in uptake or receptor content may occur with superovulation, it is apparent that these changes are probably not severe enough to disturb ovum normality (Seidel, 1981). The changes apparently do not prevent normal implantation and post-implantation development as superpregnancy normally ensues. In fact, litter sizes in hamsters have been reported as large as 27 (Fleming and Yanagi- machi, 1980). Therefore, steroids may rather play a role in effecting developmental changes of early preimplantation embryos and cleavage. However, nearing implantation, embryos possess aromatase activity and are probably able to synthesize their own steroids (Dickmann and Dey 1974; Shutt and Lopata, 1981; Sengupta e:_gl,, 1981; Gadsby g:_al,, 1981; Hoversland g:_gl,, 1982; Wu and Lin, 1982b), and thereby reduce their uptake accordingly. This may be the case for hamster embryos at the morula stage in the present study. The limited studies on oxygen consumption and 14 CO2 production exhibit rates several-fold higher than reports in the literature for mouse, rat, rabbit and rhesus monkey ova (which are extremely varied) (Fridhandler, 1961; Mills and Brinster, 1967; Brinster, 1971b; Magnus- son e:_al,, 1977). This may be due to the hormonal stimulation ad- ministered to our hamsters and monkeys to induce ovulation. It has been demonstrated that oxygen consumption increases with HCG-induced maturation in rat oocytes (Magnusson §:_al., 1981). Also, experiments with rhesus monkeys used ova collected at laparotomy, without ovula- tion induction, from a heterogeneous population, and were not assessed 52 for viability prior to culture (Brinster, 1971b). Although the volume of the squirrel monkey occyte is 3-4 times that of the hamster ovum, the two were equivalent in their rates of oxygen consumption. This is contrary to previous measurements on rabbit and mouse embryos (Mills and Brinster, 1967), which were more direct. It is possible that either the sensitivities of the techniques differ, or that the mature hamster ovum maintains a higher metabolic activity than the immature primate oocyte. This may compensate for the smaller size of the hamster ovum. The mouse ovum, in fact, possesses LDH activity 80 times that of other mammalian oocytes, including the squirrel monkey (Brinster, 1967b). The ratio of CO2 production to 02 consumption of 0.19 for oocytes from squirrel monkeys was roughly 9—fold greater than that calculated for the unfertilized mouse oocyte (Brinster, 1967a), but still quite depressed. It appears that glucose oxidation is extremely low in early embryonic development. In the mouse oocyte, less than 5% of oxygen uptake is due to glucose oxidation, with the rest primarily due to pyruvate (Brinster, 1969). The 2-deoxygluc0se experiments open up an exciting area of quick assessments of ovum metabolism and viability. Degenerate oocytes from squirrel monkeys showed a greatly diminished uptake of 2-DG compared to oocytes assessed as viable by vital dye assays. The uptake of 2-DG by viable oocytes incubated with or without insulin was determined. Although immunologic cross-reactivity between insulin of New World primates and cattle is low, the hypoglycemic action of bovine insulin is still effective in most primates (Howard, 1983). Insulin normally enhances glucose uptake by most body tissues. Yet, pharmacologic 53 concentrations of insulin were required to significantly increase 2-DG uptake beyond controls in rat thymocytes, jg_yi:r9_(5egal and Ingbar, 1980). In our studies of early primate embryos, jg_yi:r9, 2-DG uptake was not insulin-sensitive. The lowest levels of insulin used here were still greater than 50 times the physiological levels in serum from squirrel monkeys (Davidson and Blackwell, 1968). (Insulin levels in follicular fluid from squirrel monkeys are not currently avail- able.) Therefore, insulin effects at the oolemma may not be normally operational. In fact, previous work has shown that the addition of insulin to the culture medium had no effect on either the rate of oocyte maturation or jg_yj:rg_fertilization in squirrel monkeys (Kuehl and Dukelow, 1979), or on embryonic deve10pment in the mouse (Brinster, 1965). The experiment on 2-DG uptake with jg_yi:rg_fertilization of oocytes from squirrel monkeys is consonant with the large body of data showing low utilization of glucose by early embryos of most mammals. The results of the present studies indicate that biochemical changes were detected in the primate ovum with jg_yj:r9_fertilization, including augmented incorporation of 3H-uridine and steroid uptake and diminished incorporation of 3H-leucine. The embryos are viable and follow metabolically normal development comparable with preimplanta- tion development of other mammalian species. SUMMARY AND CONCLUSIONS The present studies were designed to evaluate the viability and biochemical alterations of squirrel monkey ova fertilized and de- veloped jg_vitro. In addition, the effects of ovulatory regimens on the above variables were determined. The following conclusions resulted from the data obtained: Staining with vital dyes as an index of viability l. Exclusion of trypan blue and uptake and fluorescence with fluore- scein diacetate by ova from hamsters and squirrel monkeys was highly correlated with in vitro development and relative synthe- ses of RNA and protein. Macromolecular synthesis in the early embryo 1. 3H-Uridine incorporation and uptake by squirrel monkey oocytes were reduced at 36 hr after HCG administration compared to 16 hr. Relative incorporation and uptake of 3H-uridine both increased with embryonic development in the hamster. Superovulation had no effect on either variable during embryonic development in the hamster. 3H-Uridine incorporation increased at jg yi:rg_fertilization in squirrel monkey ova. Another increase occurred at the second cleavage division. 54 55 3H-Leucine incorporation decreased with ovum maturation, and thereafter remained constant to the two-cell stage, fertilized jg_ vitro. Steroid uptake by the early embryo l. Uptake of both estradiol-17B and progesterone by embryos re- covered from superovulated hamsters increased with embryonic development. The superovulatory regimen reduced uptake of both steroids at virtually all stages analyzed. Uptake of both steroids was increased with fertilization ig_y1:rg_ and first cleavage in the squirrel monkey. Only the progesterone increase was statistically significant. Changing the ovulatory regimen for squirrel monkeys from 4 to 5 days of FSH prior to HCG administration had no appreciable affect on steroid uptake. Metabolism of the ovum and early embryo 1. Oxygen consumption by immature oocytes from squirrel monkeys was similar to mature hamster ova. Unfertilized squirrel monkey oocytes incorporated 4l.9 picograms of glucose carbon over a 4 hr period. f 14 Utilization of the above variables produced a molar ratio 0 CO 2 production from glucose to total oxygen consumed estimated at 0.19. The uptake of 2-deoxyglucose by unfertilized oocytes from squirrel monkeys was not altered by the addition of insulin. There was no change of 2-deoxyglucose uptake at jg_vitro fertili- zation in squirrel monkeys. 56 2-Deoxyglucose may be used as a viability indicator of primate 0V6. 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APPENDICES APPENDIX A BIOCHEMICAL EVALUATION OF THE UNFERTILIZED HUMAN OOCYTE* A pilot study was designed to investigate the effect of the time of oocyte recovery on 3H-uridine incorporation. Due to the difficulty in obtaining patients and oocytes, only two oocytes were processed. Protocol Patients were administered 5000 I.U. HCG on day 12 of the men- strual cycle to induce ovulation. Laparoscopic procedures and egg collection were routine (Jones g: al,, 1982). Incubation and pro- cessing of human eggs was as described for oocytes from squirrel monkeys. Data Morphologic data on the human oocyte recoveries are recounted in Table l and Figure l. Incorporation of 3H-uridine was reduced by 35 hr following HCG administration, compared to 12 hr after (Table 2). References Jones, H.W., Jones, G.S., Andrews, N.C., Acosta, A., Bundren, C., Garcia, J., Sandow, 8., Veek, L., Wikes, C., Witmeyer, J., Wortham, J.E. and Wright, G. (1982). The program for jg_vitro fertilization at Norfolk. Fertil. Steril. 38, 14-21. *This work was approved by the Human Subjects Committee of Michigan State University, East Lansing and Edward W. Sparrow Hospital, Lansing, Michigan. 70 71 TABLE 1 Morphologic Data on Human Oocyte Recoveries Patient N0. Variable 002 005 Ovary punctured Right Left Follicle size by ultra- =20 mm =20 mm sound scan Volume of follicular «5.81 4.4 fluid Compactness of cumulus Loose Loose cell mass Oocyte Morphology Oolemma pulled away Ovum started to from zona pellucida; shrink; expanded ovum expanded in in cultured culture Meiotic state Germinal vesicle Germinal vesicle intact broken down 1Follicular fluid was combined with the saline used to flush the cannula prior to volume measurements. 72 Figure 1. Photograph of a human oocyte recovered 12 hr after HCG administration. Note the intact germinal vesicle and nucleolus. 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