. w...‘l.er~.7\!§ 11144. . . s..71111..1.1.l§)3in€.§l\ .duIJ-udlnungi fl.” W‘WT LIBRARY Michigan §tate University i n . l ,1. This is to certify that the thesis entitled Culture variates affecting in vitro fertilization of squirrel monkey (Saimiri sciureus) oocytes. presented by Philip J. Chan has been accepted towards fulfillment of the requirements for Ph.D. Physiology degree- in w QMMLV Major professor Date July 22,1983 0-7639 MSUis an Arm-"1"“- ‘ ' '1, '"n. ‘, Institution 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. no Nor (Jami R .EI 7'! M ”55 0va ‘33.? 57‘ 220$; CULTURE VARIATES AFFECTING IN VITRO FERTILIZATION OF SQUIRREL MONKEY (SAIMIRI SCIUREUS) OOCYTES By Philip James Chan 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 CULTURE VARIATES AFFECTING IN VITRO FERTILIZATION OF SQUIRREL MONKEY (SAIMIRI SCIUREUS) OOCYTES By Philip J. Chan These studies were conducted to investigate the effect of energy substrates (lactate, pyruvate and glucose), human cord serum and hypotaurine on the maturation and fertilization of squirrel monkey oocytes in vitro. Studies were also done to examine the effects of follicular fluid and calcium on the motility of squirrel monkey sperm cells. ' Oocyte maturation was higher in cultures containing 5.6 mM glucose (111/303, 36.6%) than in cultures without glucose (12/82, 14.6%). Lactate (50 mM) enhanced oocyte maturation in the presence of glucose (5.6 mM). Human cord serum treatment enhanced oocyte maturation (17/35, 48.6%) above fetal calf serum treatment (14/56, 25.0%). The addition of hypotaurine (0.5 mM) to the culture medium did not affect oocyte maturation. Squirrel monkey sperm cells showed enhanced motility (compared with the control) in culture media containing .pyruvate (1.05 mM), human cord serum (20%), hypotaurine (0.5 mM), follicular fluid and calcium (7.1 mM). Glucose supplementation (5.6 mM) had no effect on ferti— lization of oocytes in vitro. However, in the presence of glucose, pyruvate (1.05 mM) enhanced fertilization . In the absence of glucose, lactate (50 mM) but not pyruvate, enhanced fertilization. Human cord serum (20%) and hypotaurine (0.5 mM) treatments did not enhance fertilization. Fertilized oocytes did not cleave in the absence of glucose (5.6 mM). Lactate (25 and 50 mM) and pyruvate (0.25, 0.5 and 1.05 mM) supplementation in culture medium had no effect on first cleavage of fertilized oocytes. No differences in first cleavage were observed in cultures containing human cord serum or hypotaurine when compared with the control. ACKNOWLEDGMENTS I wish to extend very special thanks to Dr. w. Richard Dukelow for his encouragement and support on scientific ideas and research at the Endocrine Research Unit. The valuable lessons I have learned will remain with me through the years ahead. I wish to thank members of my advisory committee, Dr. E.M Rivera, Dr. N.E. Robinson, Dr. H.A. Tucker and Dr. L.F. Wolterink for critically evaluating this work, making it and future endeavors more valuable. I also wish to thank everyone at the Endocrine Research Unit for their friendship and cooperative research efforts. My warmest appreciation goes to my parents, Mr. and Mrs. Chan Hon Yew, my brother and sister, all whom I miss the last seven years, for their understanding and support through the tough years. Finally, I wish to give a standing ovation of thanks to Hilda, my better half, who shared the depths of disappointment and the delights of success with me. ii TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . Overview of mammalian fertilization. Sperm cell capacitation and acrosome reaction. The process of oocyte maturation . . Sperm cell binding on the oocyte zona pellucida. The process of fertilization . . . . Variables affecting the motility of sperm cells in culture . . . . . . . Energy substrate utilization by gametes. . . Human cord serum as a culture medium supplement. . . The effect of hypotaurine on sperm cells . . Role of calcium ions in sperm cells. . . The role of follicular fluid in sperm cell motility. Variables involved in oocyte maturation and in vitro fertilization . . . . Energy substrate metabolism by oocytes and embryos in culture. . . The development of embryos in human cord serum . . The effect of hypotaurine on in vitro fertilization. MATERIALS AND METHODS . . . . . . . . . . Experimental animals . . . . Follicular induction regimen . . Laparoscopic recovery of squirrel monkey oocytes Semen collection . . Culture media. . Criteria of maturation and fertilization Triple— staining of squirrel monkey sperm cells Statistical analysis of data . . . . . . . . Experimental design. . . . . . . . . . . . . . . iii Page vi vii 0(3)wa U) H Page RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . 37 In vitro maturation of squirrel monkey oocytes . . . . 37 I. Effect of pyruvate and lactate supplementation on oocyte maturation in a glucose—containing medium. . . . . . . . . 37 II. Pyruvate and lactate supplementation effects on on oocyte maturation in a glucose- -free medium . 37 III. Effect of human cord serum on oocyte maturation 39 IV. Effect of hypotaurine on oocyte maturation. . 42 V. Effect of time of HCG administration on oocyte maturation. . . . . 42 Variables involved in squirrel monkey sperm cell motility. . . . . . . . 45 I. Effect of pyruvate and. lactate on sperm cell motility. . . . . . . . . . . . . . . . . 45 II. Effect of human cord serum on sperm cell motility. . . . . . . . . . . . . . . . 45 III. Effect of hypotaurine on cell motility. 48 IV. Effect of calcium on sperm cell motility. 48 V. Effect of follicular fluid on sperm cell motility. . 50 VI. Effect of dbcAMP on the sperm acrosome reaction 53 In vitro fertilization of squirrel monkey oocytes. 55 I. Effect of pyruvate and lactate on in vitro fertilization in glucose-containing medium. 55 II. Effect of pyruvate and lactate on in vitro fertilization in a glucose—free medium. 55 III. Effect of human cord serum on in vitro fertilization . . 58 IV. Effect of hypotaurine on in vitro fertilization 58 V. Effect of time of HCG administration on in vitro fertilization of matured oocytes. . . . 58 Cleavage of in vitro fertilized squirrel monkey oocytes . . . ~. 59 I. Effect of pyruvate and lactate on first cleavage in glucose- containing medium . 59 II. Effect of pyruvate and lactate on first cleavage in glucose- -free medium . . . . . . 59 III. The effect of human cord serum on first cleavage of fertilized squirrel monkey oocytes 59 IV. The effect of 0. 5 mM hypotaurine on first cleavage of in vitro fertilized squirrel monkey oocytes. . . . . . . . . 61 DISCUSSION. l O O I C O O I I D O O I O O O O O O I O I D I 62 Oocyte maturation. . . . . . . . . . . . . . . . . 62 Motility of sperm cells. . . . . . . . . . . . . . . . 64 In vitro fertilization . . . . . . . . . . . . . . 68 Cleavage of fertilized oocytes . . . . . . . . . . . 70 SUMMARY AND CONCLUS I ONS C C I O C C O O I I I O I O O O O O 7 1 REFERENCES. . . . . . . . . 73 APPENDICES I. ANALYSIS OF VARIANCE (TWO WAY) . . . . . . . . . . 98 II. A THREE YEAR OBSERVATION OF SQUIRREL MONKEY OOCYTE MATURATION, FERTILIZATION, CLEAVAGE AND DEGENERATION C O O O C C O O 0 O D I O h 0 I O O 101 III. PUBLICATIONS BY THE AUTHOR . . . . . . . . . . . . 105 IV. ABSTRACTS BY THE AUTHOR. . . . . . . . . . . . . . 107 VITA. o o a a a o a u A o o o o o o o o TABLE 10. 11. 12. 13. LIST OF TABLES Effect of pyruvate and lactate on the in vitro maturation of squirrel monkey oocytes in glucose- containing medium . . . . . . . . . The effect of pyruvate and lactate on the in vitro maturation of squirrel monkey oocytes in glucose— free medium . . . . . . . . . . . . . . . . . . . Effects of supplementation with human cord serum on oocyte maturation and fertilization . . . . . . . . . Effect of 0. 5 mM hypotaurine on oocyte maturation and fertilization . . . . . . . . . . . . . The effect of HCG injected together or separately from the final FSH dose on squirrel monkey in vitro oocyte maturation and fertilization . . . . . . . Effect of pyruvate and lactate on fertilization of squirrel monkey oocytes in glucose-containing medium. The effect of pyruvate and lactate on in vitro fertilization of squirrel monkey oocytes in glucose— free medium . . . . . . . Effect of pyruvate and lactate on cleavage of squirrel monkey oocytes in glucose-containing medium. Effect of pyruvate and lactate on oocyte maturation in a glucose— containing medium (ANOVA). . . . Effect of pyruvate and lactate on oocyte maturation in a glucose— —free medium (ANOVA). . . . . . . . . Effect of pyruvate and lactate on in vitro fertili— zation in a glucose—containing medium (ANOVA) . Effect of pyruvate and lactate on in vitro fertili— zation in a glucose—free medium (ANOVA) . . . . . . Effbnr of pyruvate and lactate on first cleavage in a glucose— —containing medium (ANOVA) . . . . . vi Page 38 40 41 43 44 56 57 60 98 98 99 99 100 FIGURE 10. 11. 12. 13. 14. LIST OF FIGURES Diagramatic representation of the acrosome reaction . The successive stages of fertilization in the hamster Laparoscopic procedure for recovery of squirrel monkey oocytes. . . . . . . . . . . . . . . . Four staining patterns observed after staining sperm cells with the triple stain . . . . . . . . . . . Energy substrate supplementation effects on squirrel monkey sperm motility . . . . . . . . . . . . . Differential effects of human cord serum and fetal calf serum on the motility of squirrel monkey sperm Cells C O O C O I I I C O O O C O O O The effect of hypotaurine, taurine and epinephrine on squirrel monkey sperm motility . . . . . . . The effect of calcium on squirrel monkey sperm cell motility. . . . . . . . . . . . . . . . . . . . . The effect of follicular fluid on squirrel monkey sperm cell motility . . . . . Effect of dbcAMP on the acrosome reaction of squirrel monkey sperm cells. . . . . . . . . . . . . . . . . A three year study of squirrel monkey oocyte in vitro maturation. . . . . . . . . . . . A three year study of squirrel monkey in vitro fertilization . . . . . . . . . . . . A three year study of first cleavage of in vitro fertilized squirrel monkey oocytes. . . . . . A three year study of squirrel monkey oocyte degeneration. . . . . . . . . . vii Page 10 28 34 46 47 49 51 52 54 101 102 103 104 INTRODUCTION The words ”in vitro", as defined by Dorland's Medical Dictionary, mean "within a glass”. Indeed, the use of an in vitro system to culture gametes of laboratory species, domestic animals and primates, have contributed to a significant understanding of reproductive physiology. The transfer of in vitro fertilized embryos to recipient females have produced viable offspring, by—passing hurdles that may have made it impossible for some females to conceive. The advantages of an in vitro system are the convenience of observing and manipulating the developing embryo, and the availability of embryos at a particular stage of development for biochemical, chromosomal assays and electron microscopic analysis. There are several published reviews on research in oocyte maturation and in vitro fertilization (Chang, 1968; Thibault, 1969; Brackett, 1981, 1983; Kennedy, 1972; Edwards and Steptoe, 1975; Kuehl, 1976; Bavister, 1980; Mastroianni and Biggers, 1981; Hafez and Semm, 1982). The present research uses a squirrel monkey in vitro fertilization technique to examine the effects of naturally—occurring compounds and fluids on fertilization and embryo development. A squirrel monkey in vitro fertilization system has been used at the Endocrine Research Unit for a number of studies. These studies demonstrated that cumulus cells were necessary for oocyte maturation, that caffeine and dibutyryl cyclic adenosine monophosphate (dbcAMP) stimulated squirrel monkey sperm motility, and that addition of dbcAMP to the culture system increased the percentage of in vitro fertilized oocytes by about 50% (Chan, 1981). The squirrel monkey provides a model for in vitro fertilization studies in a species phylogenetically related to the human. The objectives of the present studies were: (1) to test the hypothesis that lactate, pyruvate, glucose, cord serum and hypotaurine play a role in the maturation of squirrel monkey oocytes in vitro. (2) to test the hypothesis that calcium, follicular fluid, and the compounds listed in objective 1, affect the I motility of squirrel monkey sperm cells. (3) to test the hypothesis that dbcAMP accelerates the acrosome reaction. (4) to test the hypothesis that the compounds, listed in objective 1, affect in vitro fertilization and cleavage of squirrel monkey oocytes. The rationale behind these studies was that if one could determine the factors that play a physiological role in the fertilization process of squirrel monkey oocytes, this information could be used to increase the efficiency of the in vitro fertilization procedure. CULTURE VARIATES AFFECTING IN VITRO FERTILIZATION OF SQUIRREL MONKEY (SAIMIRI SCIUREUS) OOCYTES LITERATURE REVIEW Overview of mammalian fertilization Fertilization is defined as the union of the sperm cell and the oocyte. Both the sperm cell and the oocyte must be mature. This means that the sperm cell must be capacitated and acrosome—reacted (explained on pages 4 and 5) and the oocyte must be in the haploid state (22 chromosomes in the squirrel monkey) at the metaphase of the second meiosis. The following is a brief description of the maturation process of the sperm cell and the oocyte. Sperm cell capacitation and acrosome reaction In the testis, the spermatogonia differentiate into the sperm cell (also called the spermatozoa). The sperm cell undergoes maturation in the epididymis (Bedford, 1979). Epididymal maturation is the process whereby sperm Cell components undergo biochemical and physiological changes (for details, see Orgebin—Crist, 1969; Bedford, 1975; Hamilton, 1977). The sperm cell also acquires motility (Mohri and Yanagimachi, 1980). At the same time, the sperm plasma membrane adsorbs a variety of substances (glycoproteins, sialic acid, antigens, carnitine, glycerylphosphoryl choline and lipids) from the epididymal fluid (Yanagimachi, 1981). Upon ejaculation, the sperm cell surface is further coated with substances (lactoferrin, phospholipids, lipoproteins, decapacitation factor) from the seminal plasma (Oliphant and Singhas, 1979). In the female genital tract (or in a culture system) the sperm cell undergoes capacitation, which means that the sperm cell acquires the capacity to fertilize the oocyte. Capacitation involves the removal or alteration of the coating materials on the sperm cell surface (Weinman and Williams, 1964; Pikd, 1967, 1969; Hunter and Nornes, 1969; Bedford, 1970; Johnson, 1975) over a period of time (capacitation time). The capacitation time of squirrel monkey sperm cell is 2 to 5 hours (Kuehl and Dukelow, 1982) and is similar to the time required by the human sperm cell. Factors that enhance the initiation and rate of capacitation in vivo or in vitro include high ionic strength medium (380 mOSM; Brackett and Oliphant, 1975), cumulus cells (Gwatkin et al., 1972; Gwatkin, 1977) and hypotaurine (Liebfried and Bavister, 1982). The sperm cells are transported (for details, see Bishop, 1961, 1969; Blandau, 1969; Bedford, 1970, 1972; Thibault, 1972, 1973; Zamboni, 1972; Blandau and Gaddum-Rosse, 1974; Hafez and Thibault, 1975; Overstreet and Katz, 1977; Overstreet and Cooper, 1978, 1979; Overstreet 95 al., 1978; Shalgi and Kraicer, 1978; Cooper gt al., 1979; Hunter, 1975, 1980) from the site of semen deposition to the ampullar- isthmal junction of the oviducts where the sperm cells interact with the oocyte(s). The next obligatory step in the maturation of the sperm cell prior to oocyte penetration is the acrosome reaction. The acrosome reaction (see Figure 1) is defined as the fusion and vesiculation of the outer acrosomal membrane and plasma membrane of the sperm (Dan, 1956; Barros gt at., 1976), and the activation of sperm cell acrosomal enzymes (hyaluronidase, esterase, neuraminidase, acrosin) located within the matrix of the acrosomal cap (Mancini et al., 1964; McRorie and Williams, 1974; Gould and Bernstein, 1975; Bryant and Unnithan, 1973; Green and Hockaday, 1978). The physiological function of the acrosome reaction is to release acrosomal enzymes that help the sperm cell penetrate the oocyte investments (cumulus cells, corona radiata and zona pellucida). The acrosome reaction also prepares the sperm plasma membrane for fusion with the oocyte plasma membrane (Yanagimachi and Noda, 1970; Yanagimachi, 1977). The factors involved in initiating the acrosome reaction are cumulus cells (Austin and Bishop, 1958; Yanagimachi and Noda, 1970), calcium ions (Yanagimachi and Usui, 1974; Roomans, 1975; Garbers and Kopf, 1980), pH of the medium (Pavlok, 1968; Miyamoto 25 al., 1974; Hyne and Garbers, 1980), pyruvate and glucose (Rogers and Yanagimachi, 1975; Hoppe, Sperm cell Plasma membrane Acrosome [,0uter acrosomal membrane Acrosomal cap Postacrosomal region Figure 1. Diagramatic representation of the acrosome reaction: (1) Before the acrosome reaction (2) Fusion of plasma and outer acrosomal membrane (3) Release of acrosomal enzymes (4) The reaction is complete. (Bedford, 1967). 1976), albumin (Lui and Meizel, 1977; Davis, 1978) and catecholamines (Cornett and Meizel, 1978). The sperm cell that has gone through capacitation and the acrosome reaction stages is now ready to penetrate the mature oocyte. The maturation of an oocyte is described in the next section. The process of oocyte maturation Oocytes are derived from oogonia in the female ovary through a process called oogenesis (for details, see Berrill and Karp, 1976). During the fetal life of an animal, the oocytes are arrested at the diplotene stage of prophase I (Brambell, 1960; Franchi st 31., 1973). The diplotene stage is characterized by decondensed chromosomes in the oocytel nucleus (germinal vesicle). The inhibitory signal (oocyte maturation inhibitor, OMI) has been suggested to come from the granulosa cells around the oocyte (Tsafiri and Channing, 1975; Hillensjd 33 al., 1978, 1980). Resumption of oocyte meiosis can be triggered by an injection of luteinizing hormone (LH) into the animal or by the LH surge prior to ovulation (Edwards, 1965) or by releasing the oocyte from the follicle (Magnusson, 1980). The oocyte proceeds through metaphase I (germinal vesicle breakdown) and extrudes the first polar body. The second meiotic division occurs next and progresses to metaphase II. It is at this metaphase II stage that the oocytes are considered mature and ready for sperm penetration. In vivo, the mature oocytes are ovulated and await sperm cells at the ampulla isthmal junction of the oviducts. Oocytes recovered from the follicles of the ovary by laparoscopic techniques (used in the present studies) may or may not be matured. Immature oocytes are not penetrable by sperm cells (Iwamatsu and Chang, 1972; Barros and Munoz, 1973, 1974; Niwa and Chang, 1975). Immature oocytes are matured in vitro by incubating them for a period of time in culture medium. Variables affecting oocyte maturation are the presence of cumulus cells (Donahue and Stern, 1968; Kennedy and Donahue, 1969; Cross and Brinster, 1970; Cross, 1973; Binor and Wolf, 1979; Magnusson, 1980; Fukui and Sakuma, 1980), cyclic AMP (Racowsky, 1983), cyclic GMP (Kostellow and Morrill, 1980), estrogen and progesterone (Smith and Tenney, 1980; Rice and McGaughey, 1981), estrous cycle stage (Nekola, 1980), ovarian glycosaminoglycans (Eppig, 1981), prostaglandins E2 and F2“ (Lemaire Et al., 1973; Mandelbaum 35 21., 1982) and the age of the animal (Peluso and Hutz, 1980). Sperm cell binding on the oocyte zona pellucida The surface of the zona pellucida contains sperm receptor sites with a high affinity for sperm cells (Yanagimachi, 1981). The receptor is a glycoprotein, designated ZP3 (Bleil and Wassarman, 1980). The attachment of sperm cells to ZP3 (molecular weight 83,000) requires calcium ions (Saling gg gl., 1978; Heffner gg gl., 1980). Acrosome—reacted sperm cells expose a gelatinous matrix located in the inner acrosomal membrane (Yanagimachi, 1977) that enables the sperm cells to attach to the zona pellucida (Huang gg gl., 1981). In addition, there are zona receptor sites on the sperm surface that are involved in the sperm—zona pellucida interaction (Gwatkin and Williams, 1977; Yanagimachi gg gl., 1981). It is believed that these zona receptor sites confer the species—specificity (that is, sperm cells of one species will not attach to the oocytes of another species) to the sperm cells. The process of fertilization The sperm cell that is attached to the zona pellucida quickly penetrates the zona (see Figure 2) by means of zona lysins (acrosin and SS—reductase) released through the acrosome reaction (Austin and Bishop, 1958; Bedford, 1974; Niya and Yanagimachi, 1976; Zaneveld, 1976) and attaches to the oocyte plasma membrane (Yanagimachi, 1966? Yang gg gl., 1972). A block to polyspermy (fertilization by more than one sperm cell) is established within 5 — 15 min of sperm attachment to the oocyte plasma membrane (for details, see Austin and Bishop, 1957; thtos and chagimachi, 1972; Sato, 1979). Basically, attachment of the sperm results in mobilization of calcium ions through the oocyte triggering 10 Zona pellucida 2. First polar body Cortical granules Plasma perivitelline space membrane Sperm tail remnants Second polar body Pronuclei Figure 2. The successive stages of fertilization in the hamster. 1.Sperm cell penetrating zona pellucida; 2.Sperm cell attaching to oocyte plasma membrane; 3. Decondensation of sperm head; 4. Extrusion of the second polar body; 5. Formation of pronuclei; 6. The completion of fertilization. (Yanagimachi, 1981). 11 cortical granules to release proteases (Barros and Yanagimachi, 1971; Gwatkin, 1977; Wiuf and Hamada, 1977) and an ovoperoxidase (Gulyas and Schmell, 1980; Schmell and Gulyas, 1980) which alter the sperm receptor sites on the zona pellucida, as well as causing the zona pellucida to harden and block further sperm cell entry into the oocyte. The attachment of the sperm cell to the oocyte plasma membrane also triggers a plasma membrane block to polyspermy. Unlike the zona reaction which involves enzymes, and ovoper— oxidase, this block is electrically-mediated (Wiuf g5 gl., 1979; Okamoto gg g£., 1977; Hagiwara and Jaffe, 1979). Details of the mechanism remain unknown. The oocyte plasma membrane is covered with microvilli that constantly disappear and reappear (Shalgi and Phillips, 1980). Sperm cell plasma membrane at the postacrosomal or equatorial region fuses with the oocyte microvilli (Yvnagimachi and Noda; 1970) and the sperm head decondenses. The sperm tail is gradually incorporated into the oocyte plasma membrane. Developing sperm and oocyte pronuclei are observed about 2 hours after sperm cell entry. The two pronuclei are drawn together at the center of the oocyte, the nuclear envelopes are disassembled and the chromosomes mix (syngamy) as the first mitotic division takes place (Longo and Anderson, 1969; Condos gg gl., 1972; Zamboni, 1972; Longo, 1973; Anderson gg gl., 1975). This completes the process of fertilization. 12 Variables affecting the motility of sperm cells in culture The motility of sperm cells constitute the only mechanism by which sperm cells penetrate the zona pellucida to fuse with the oocyte plasma membrane (Pincus, 1930; MacLeod and Gold, 1953; Yanagimachi and Noda, 1970; Bedford, 1974; Noda and Yanagimachi, 1976; Yanagimachi, 1981). Sperm cells cultured in artificial media prior to in vitro fertilization, lose their motility after a very short incubation period. This is a problem in in vitro fertilization because the sperm cells must be kept motile for long periods to allow enough time for oocytes to mature in culture and for the sperm cells to penetrate the matured oocytes. As explained in the previous sebtion, sperm cells are unable to penetrate immature oocytes (see page 8). It is important, then, to determine the physiological variables that sustain sperm cell motility in the female reproductive tract and in vitro. Variables that influence sperm cell capacitation or the acrosome reaction are equally important. Capacitation and the acrosome reaction are obligatory steps that lead to the fertilization of an oocyte and hence, the variables that promote these two steps have been studied. Some of these variables are: concentration and type of metabolic energy substrate, presence or absence of cumulus cells, follicular fluid, calcium levels, oxygen tension, presence of cyclic nucleotides, presence of beta 13 amino acids and temperature. I have examined the first of the variables listed above; the energy substrates. Energy substrate utilization by gametes A variety of metabolic substrates are utilized by sperm cells of different species, including fructose, glucose and pyruvate (Eliasson EE,§£°’ 1968; Voglmayr gg ii" 1970; Rogers E£.El's 1981; Leese gg‘gl., 1981). Enzymes involved in the glycolytic pathway, the pentose phosphate pathWay and the Krebs cycle are present in sperm cells (Peterson and Freund, 1970). These enzymes help to metabolize energy substrates and produce adenosine triphosphate (ATP) to maintain sperm cell viability. The energy substrates found in reproductive tract fluids (Bishop, 1957) are metabolized by sperm cells and are a source of energy for the sperm cells swimming in their sojourn to the site of fertilization (Hamner and Williams, 1963). Energy substrates also affect sperm cell capacitation (Hoppe, 1976). In the in vitro environment, glucose (5.5 mM), but not fructose (5.5 mM), promote capacitation in mouse sperm cells. Pyruvate (0.23 mM) or lactate (23.3 mM) had no effect on sperm cell capacitation (Hoppe, 1976). Recent studies on mouse sperm cells (Fraser and Quinn, 1981) confirmed the necessity of glucose (5.56 mM) for motility and sperm activation. Using labelled glucose, Murdoch and White (1967) showed that the rabbit sperm cell metabolizes glucose primarily 14 through the Embden—Meyerhof (glycolysis) pathway. Sperm maintained in vitro beyond 2 hours gradually shifted meta— bolism to the pentose phosphate pathway (measured by labelled carbon dioxide produced from [1-;4Cflglucose) because of a decline in aerobic glycolysis and respiration (associated with senescence changes). Glucose was the main substrate meta— bolized by the rabbit sperm cells. Guinea pig sperm cells do not utilize glucose for energy metabolism (Rogers gg gl., 1981). In fact glucose appears to retard the initiation of the acrosome reaction (Rogers and Yanagimachi, 1975). Fructose or mannose also retards the acrosome reaction. Glucose has also been shown to inhibit oxidative processes in bull sperm cells (Lardy and Phillips, 1941). It is clear that the effects of glucose and the other energy substrates (fructose, lactate, pyruvate) are inhibitory in some species (on motility, capacitation or the acrosome reaction) and supportive in others. There have been no reports on the effects of energy substrates on squirrel monkey sperm cell motility and hence, the present studies were conducted. Human cord serum as a culture medium supplement Serum is a common component used as an additive in culture media for gametes and embryos. A dialysable heat—stable fraction in blood serum has been isolated and 15 shown to stimulate hamster sperm motility and capacitation (Yanagimachi, 1970; Morton and Chang, 1973). Bavister (1975) analysed human serum and found that the factor had a molecular weight of 100 - 200. It was proposed that this serum factor stimulated the synthesis of cyclic AMP by activating hamster sperm cell adenylate cyclase (Morton and Albagli, 1973; Morton and Chang, 1973). The elevation of CAMP activated CAMP— dependent protein kinase in the sperm cell (Hoskins gg gl., 1972; Garbers gg gl., 1973) which in turn, phosphorylated troponin which is involved in sperm cell motility (Bailey and Villar—Palasi, 1971). The serum from bulls and heifers also affects the motility and acrosome reaction of sperm cells (Senger and Saacke, 1973; Senger g5 gl., 1981). Those studies compared the effect of fresh serum and frozen—thawed serum, and the results indicated fresh serum promoted more acrosome reactions (in bull sperm cells) than frozen—thawed serum (Senger gg gl., 1981). An explanation for the difference observed in the serum has not been forthcoming. The effect of human cord serum components on the motility of the sperm cell has never been studied in any species. Several reasons prompted the initiation of studies into human cord serum: (a) the presence of 3 growth factors in the serum (Hsu, 1973; Hsu g5 g£., 1974; Hsu, 1980), and (b) the rising use of cord serum in human in vitro fertilization trials. The purpose of the experiment was to determine if there were 16 factors in cord serum capable of sustaining sperm cell motility. The effect of hypotaUrine on sperm cells In 1976, Bavister gg gl. showed that a low molecular weight factor from hamster adrenal gland extract stimulated hamster Sperm cell motility and the acrosome reaction. It has been suggested that this factor is the same factor found in serum responsible for enhancedsperm cell motility (Bavister and Yanagimachi, 1977). It was postulated that the factor was epinephrine (Cornett and Meizel, 1978; Cornett et al., 1979; Meizel and Working, 1980), but researchers were unable to stimulate sperm cell motility with epinephrine. Mrsny gg g£., (1979) found high levels of taurine in the adrenal gland extract and successfully stimulated sperm cell motility with taurine. Meizel 2E.El°’ (1980)reported finding a second related compound, hypotaurine, in the adrenal gland extract. Hypotaurine also stimulated hamster sperm cell motility. In addition, they found taurine (at a concentration between 0.05 — 0.12 mM) and hypotaurine (at 0.46 - 0.47 mM) in bovine follicular fluid. These compounds are also present (0.054 — 1.525 mM) in rabbit uterine and oviductal fluids, bovine follicular fluid, and macaque (Macaca fascicularis) oviductal fluid. Taurine and hypotaurine, in combination with epinephrine, resulted in even greater sperm cell motility (Cornett gg gl., 17 1979) with whiplash flagellar movement, characteristic of capacitated sperm (Yanagimachi, 1970). Meizel and Working (1980) postulated that taurine, hypotaurine and epinephrine might inhibit phosphodiesterase (PDE) activity or, act as a chelator of metal ions, indirectly stimulating ATPase activity with consequential activation of processes leading to sperm cell capacitation. The ability of these compounds to activate the hamster sperm cell stimulated interest on identifying a similar phenomenon in primate sperm cells. The effect of hypotaurine, taurine and epinephrine on the motility of squirrel monkey sperm cells was therefore examined in the present studies. Role of calcium ions in sperm cells Calcium ions regulate structure and function of membranes and metabolic pathways (Comar, 1969) and play a vital role in sperm acrosome reaction induction (Yanagimachi and Usui, 1974), sperm binding to the zona pellucida (Saling gE gl., 1978; Heffner gg gl., 1980), oocyte maturation (Guerrier gg g£., 1978) and cell division (Whitfield g5 gl., 1979). Reyes gg gl. (1978) showed that calcium was necessary in the medium for in vitro capacitation. The percentage of sperm capacitated was enhanced with the concomitant addition of dibutyryl cyclic AMP and a calcium ionophore A23187. Purified sperm plasma membranes contain high affinity binding sites for calcium ions (Peterson gg gl., 1979) and a further increase in 18 binding is seen with the addition of sodium (0.1 M) and potassium ions (0.1 M). Hyne and Garbers (1979) speculated. that the calcium ions bind to regulatory proteins which then stimulate sperm adenylate cyclase, culminating in the acrosome reaction. One characteristic of the acrosome reaction is the release of sperm cell enzymes from the acrosomal cap. Calcium ions greatly increase proteolytic activity of an acrosomal enzyme called acrosin, a hydrolytic enzyme required for sperm penetration of the oocyte (Parrish and Polakoski, 1981). Calcium ions are involved in ciliary and flagellar movements (Blum g5 gl.,1980). Blum gg gl. (1980) showed that calcium acts through calmodulin located on axonemes (on 145 and 30s dyneins) in cilia to activate ATPase. Each interaction of a calcium—calmodulin molecule with dynein ATPase results in the release of energy. It is this energy that powers the movement of the cilia. The sperm flagellum, responsible for motility, possibly uses the same mechanism found in the cilia. Indeed, high amounts of calmodulin have been purified from sperm (Garbers gg g£., 1980). The mechanism of action of calcium ions and calmodulin on sperm cell motility is not known. The effect of calcium ions on sperm cell motility appears to vary with species. Exogenous calcium ions will initiate motility in some species (hamster, bull, guinea pig) but not in others (rabbit, human) (Morton g5 gl., 1974; Morton g5 g£., 1978; Babcock gg gl., 1979; Storey, 1975; Peterson and Freund, 19 1976; Turner and Howards, 1978). In an extensive study using detergent—treated dog sperm cells, Tash and Means (1982) showed that calcium inhibited the motility of sperm cells. It was evident that it was necessary to study the effect 9'11 0 calcium ions on squirrel monkey sperm cells. The question H1 0 whether calcium ions stimulated or inhibited the motility of squirrel monkey sperm cells was therefore investigated in my studies. The role of follicular fluid in sperm cell motility Follicular fluid is a viscous, straw—colored liquid, composed partly of secretions from the follicle (granulosa) cells and partly of exudates from plasma (for reviews, see Brambell, 1960; Zachariae, 1959; Moricard, 1969; Edwards, 1974). Follicular fluid contains glucose (Lutwak-Mann, 1954), lactic acid, cholesterol and lipids (Zachariae and Jensen, 1958), plasma proteins (Shivers gg gl., 1964; Manarang—Pangan and Menge, 1971), alkaline phosphatases, transaminases and ATPase (Cerletti and Zichella, 1961; Caucig gg gl., 1971), gonadotropins (Bjersing g5 gl., 1972), estrogens, androgens and progestagens (Parkes, 1929; Short, 1964) and hyaluronic acid (Steptoe and Edwards, 1970). The physiological function of follicular fluid is to assist in the transport of the oocyte from the follicle to the oviduct (Edwards, 1974). In addition, follicular fluid 20 induces the acrosome reaction of hamster sperm cells (Yanagimachi and Chang, 1964; Barros and Austin, 1967; Gwatkin and Andersen, 1969). Yanagimachi (1979) showed that a nondialyzable labile factor was involved in the induction of the acrosome reaction. Subsequently, it was shown that this follicular fluid factor was albumin (Lui 35 al., 1977). It is postulated that albumin adsorbs fatty acids from the sperm cell plasma membrane and destabilizes the membrane resulting in the acrosome reaction (Yanagimachi, 1981). Follicular fluid aspirated from ovarian follicles of anesthetized monkeys has never been tested for its effect on sperm cells. The effect of follicular fluid on the motility of‘squirrel monkey sperm cells was examined in the present studies. Variables involved in oocyte maturation and in vitro fertilization There are many variables that can influence the maturation and fertilization of cultured oocytes. Some of the variables affecting maturation include temperature (Kruip and Vernooy, 1982), steroids and prostaglandins (Robertson and Baker, 1969; Nekola and Smith, 1974; Bae and Foote, 1975; McGaughey, 1977; Mandelbaum SE al., 1982), cyclic nucleotides (Schatz and Morrill, 1972; Kostellow and Morrill, 1980) and cumulus cells (Donahue and Stern, 1968; Cross, 1973; Bar—Ami gt al., 1967; Hillensjg, 1979; Fukui and Sakuma, 1980; Cameron 35 al., 1983). 21 Similarly there are many variables affecting fertili— zation such as genetic factors (Kaleta, 1977; Maudlin and Fraser, 1978), sperm concentration (Austin, 1948; Niwa and Chang, 1973, 1974), senescence (Blaha, 1964; Miller and Blackshaw, 1968; Maurer 35 al., 1969; Koefoed—Johnsen, 1971; Parkening and Chang, 1976), pH of environment (Bavister, 1969; Hyne and Garbers, 1981) and autoantibodies (Menge, 1971; Jones 35 al., 1975; Tsunoda and Chang, 1976; Yanagimachi 35 al., 1981). The process of fertilization occurs under a complex interactive environment. The present studies focus on the effect of several variables (energy substrate, human cord serum and hypotaurine) on oocyte maturation, fertilization and cleavage. Energy substrate metabolism by oocytes and embryos in culture Energy substrates (carbon sources) are incorporated into macromolecules of the oocyte and developing embryo. These substrates include lactate, pyruvate, glucose and metabolites of the Kreb cycle (Brinster, 1969; Wales and Whittingham, 1970, 1973; Pike and Wales, 1972; Quinn and Wales, 1973; Murdoch and Wales, 1973; Wales, 1975; Renard 3E al., 1980). The metabolism of the substrates yield energy in the form of adenosine triphosphate (ATP). For example, the oxidation of one molecule of glucose (aerobic metabolism) yields 36 ATP molecules (or 686 kcal/mol). This oxidative reaction is found in a majority of tissues (liver, kidney, skeletal muscle etc.) 22 and depends on the availability of oxygen. The oxygen tension in oviducts (the site of fertilization) is between 40 — 75 mm Hg (Mastroianni and Jones, 1965; Auerbach and Brinster, 1968). Embryos are capable of developing under low oxygen tension (Auerbach and Brinster, 1968). The effect of oxygen tension on the fertilization of squirrel monkey oocytes has been studied (Kuehl, 1976). In some types of cells (smooth muscle), glucose is metabolized to lactate (anaerobic glycolysis) with only 2 ATP molecules produced (47 kcal/mol). Thus, one point of consideration is that glucose is metabolized by different pathways under differing conditions (oxygen tension, substrate availability). Oocytes and embryos utilize different energy substrates (Brinster, 1965; Kane, 1979). Brinster (1971) demonstrated that excised monkey ovarian oocytes cultured in vitro exhibit a 15 - 20 fold greater requirement for pyruvate than glucose. Lactate dehydrogenase, an enzyme which metabolizes pyruvate to lactate with the production of reducing (NAD+) equivalents, is present early in the oocyte (Brinster, 1965 and 1967; Auerbach and Brinster, 1967). Nicotinamide adenine dinucleotide (NAD) is involved in the electron transfer process to produce energy (ATP). High ATP to ADP ratio in oocytes causes an imbalance in the redox potential and inhibits full utilization of lactate (Quinn and Wales, 1973). Glucose—6—phosphate dehydrogenase activity in the oocyte diminishes from ovulation 23 to implantation indicating that glucose utilization by the pentose phosphate pathway diminishes as the embryo develops . (Brinster, 1966). Preimplantation mouse embryos utilize pyruvate as the major energy source (Brinster, 1971). However, a higher percentage of mouse embryos develop in culture if both pyruvate and lactate are available (Brinster, 1965; Wales and Whittingham, 1973; Quinn and Wales, 1973). The dividing mouse embryo at the 8—cell stage and beyond attains the ability to metabolize glucose (Brinster and Thomson, 1966). Glucose uptake by blastocysts has been reported (Renard g£_al., 1980). Other energy sources such as oxaloacetate, malate and sudcinate and their utilization have been reported (Brinster and Thomson, 1966), but most work has focused on glucose, lactate and pyruvate as substrates in culture systems. Thus, a second major point to consider is that the type of energy substrate utilized is dependent on the stage of development of the embryos. As pointed out earlier in the sperm cell motility section, different species utilize different substrates. For this reason, and since there are also differences in substrate utilization by different cell types and at various stages of development, there was a need to establish the effects of energy substrates on squirrel monkey oocytes, in order to refine and improve culture conditions. The present studies focus on the effect of energy substrates on oocyte maturation, in vitro fertilization and cleavage. 24 The development of embryos in human cord serum The use of heat—inactivated human cord serum was first reported to be important for in vitro development of mouse embryos by Hsu (1973). Hsu 33 El. (1974) initially cultured two-cell embryos in fetal calf serum (FCS) but development stopped at the blastocyst stage. Human cord serum (HCS) was then substituted for FCS, and a factor in the H08 promoted further development to the early somite stage. The factor in HCS was further investigated and was named embryo growth and differentiation factor 3 (EGDF—B). High concentrations (20 - 30%) of H08 were required for normal- sized embryo development. EGDF—l and EGDF—Z, are macromole- cular factors found in both FCS and HCS, needed for preim- plantation embryo development (Hsu, 1980). Successful in vitro culture of blastocysts to limb bud stages was recently reported (Chen and Hsu, 1982). The researchers designed an elaborate medium which had increasing concentrations of FCS, HCS and rat serum respectively, to meet the demands of the developing embryo. Human cord serum has also been used in the culture of in vitro fertilized human embryos (Lopata 3E al., 1978; Suzuki gt al., 1981). Lopata and colleagues reported 9 oocytes fertilized out of 22 (40.9%) while Suzuki and co-workers had 5 oocytes fertilized out of 43 (11.6%). The effect of the cord serum factors on the maturation of oocytes has never been investigated. The present studies 25 examine the effects of HCS on squirrel monkey oocyte maturation and in vitro fertilization. The effect of hypotaurine on in vitro fertilization The historical account of the discovery of hypotaurine in reproductive tract fluids has been mentioned previously. Hypotaurine and taurine enhance hamster sperm cell motility, capacitation and acrosome reaction (Cornett and Meizel, 1977; Cornett EE.§£°’ 1979; Meizel and Working, 1980). Recently, the effects of hypotaurine and taurine on the fertilization of hamster oocytes were examined (Liebfried and Bavister, 1981, 1982). They found that both compounds resulted in low percentages of fertilization unless a beta agonist, isoproterenol was also present. To date, there has been no other report on the role of hypotaurine on the fertilization.of oocytes. It was thus. hypothesized that the stimulation of sperm cell motility would result in an increase in fertilization of squirrel monkey oocytes. MATERIALS AND METHODS Experimental animals The animals used were adult squirrel monkeys (Saimiri sciureus, 500 to 600 grams) of Bolivian origin (Charles River Research Primates, Port Washington, New York) housed indoors on a 12:12 hour light:dark cycle at 210i 3°C. During the summer months (June to October), the animals were housed in gang cages outdoors (Jarosz and Dukelow, 1976). The diet consisted of a commercial high protein monkey feed (Ralston— Purina), and slices of apples daily with fresh water ad libidum. Since the female squirrel monkey does not menstruate and it is difficult to ascertain the exact day of the cycle, the hormonal administration schedule used to induce follicular growth and oocyte maturation was arbitrarily started at either 5 or 6 days prior to the time scheduled for laparoscopic oocyte recovery. This is in accord with the procedure used by other workers using the squirrel monkey (Bennett, 1967; Dukelow, 1970; Could gt at., 1973). Follicular induction regimen The follicular induction regimen consisted of four daily intramuscular (i.m.) injections of follicle stimulating 26 27 hormone (1 mg, FSH-EEK Burns—Biotec Labs., Inc., Omaha, Nebraska) and a single i.m. injection of human chorionic gonadotropin (250 IU, HCG, APL Ayerst Laboratories, Montreal), given 10 hours after the final FSH injection, on the fourth day (Dukelow, 1970). During the nonbreeding season (July through September) five days of FSH injections were used to induce follicular growth based on previously reported studies with this colony (Kuehl and Dukelow, 1975). .The hypothesis was tested that administration of the HCG concurrent with the final FSH injection would not affect oocyte maturation and fertilization, compared to giving the HCG 10 hours after the final FSH injection. This study involved 16 trials and a total of 53 animals. The results (to be discussed later, Tablefi, page 44) confirmed the hypothesis and therefore in subsequent trials HCG and final FSH injections were given concurrently. Laparoscopic recovery of squirrel monkey oocytes The laparoscopic procedure for oocyte recovery has been previously described (Dukelow 3E EL., 1971; Dukelow & Ariga, 1976). Basically this procedure consisted of anesthetizing the monkey with sodium pentobarbital (15 mg/6—7OO gm animal) 15-16 hours after the HCG injection. A small midline incision was made With a scapel and the trocar-cannula inserted through the incision and abdominal wall (Figure 3). The trocar was then removed and the.laparoscope (4 mm diameter, Karl Storz 28 Storz Laparoscope Fimbriae covering ovary 25 gauge needle used in aspirating follicular oocytes Bladder Follicle on right ovary Left ovary Figure3 . Laparoscopic procedure for recovery of squirrel monkey oocytes. 29 Co., West Germany) inserted. The abdominal cavity was insufflated with C02 passed through the cannula. A 25 gauge. needle, fitted on a 1 ml tuberculin syringe, was used to move the fimbria aside to expose the ovaries. The ovarian follicles were punctured using the needle, and the oocytes aspirated into 0.05 ml of culture medium (see page 30). The oocytes were then incubated in sterile 8—chamber tissue culture slides (Lab—Tek Products, Napierville, Illinois) at 370C in a moist atmosphere of 5% C02 in air. The oocytes in the cultures were observed through an inverted microscope every 24 hours. A culture slide incubator (Clinical Scien— tific Equipment Co., Melrose Park, Illinois) mounted on the microscope stage kept the cultures at 370C during observa— tions. Cultures contaminated with red blood cells were washed by flushing an additional 0.2 ml of culture medium into each chamber. The oocytes, being heavier, sunk to the bottom of the culture chamber and 0.2 ml of the diluted culture medium was then aspirated out carefully and discarded. The final volume in all chambers was adjusted to 0.25 ml. Semen collection The adult male squirrel monkey was held in a V—shaped restrainer (Kuehl and Dukelow, 1974) and short pulses of current (120 pulses per second at 5 msec duration) were delivered to a probe placed just inside the rectum. The voltage was gradually increased and decreased in a rhythmic 30 fashion every 3 seconds. The ejaculated coagulum plug containing the sperm cells was collected and diluted in culture medium (see below) and held at 37°C for 5—10 minutes. The oocytes in each culture chamber were then inseminated with 0.05 ml of the sperm cell suspension at 20 to 21 hours after recovering the oocytes from the ovarian follicles (105 to 106 sperm/ml). Sperm cell motility was visually assessed from the remaining aliquots of sperm cell suspension in either control medium or medium containing test compounds. The culture chamber slide containing the sperm was mounted on the inverted microscope and sperm cell motility estimations made. The percentage of motile sperm cells was visually assessed at 5 different locations in each treatment chamber and the mean and standard error of the mean recorded. Sperm cell motility assessments were made at intervals of O, 30, and 60 minutes, and then at hourly intervals up to 23 hours. In each experiment, the initial sperm cell motility, the percentage of progressive motility, the percentage of immature sperm cells (as denoted by cytoplasmic droplets on the sperm tail) and the sperm cell concentration in each chamber well were recorded. Culture media The basic medium used for culturing the oocytes and sperm cells was T0199 (with 2mM HEPES buffer, Earl's salts, 31 L—Glutamine, 5.6 mM D-glucose, catalog # 380—2340, GIBCO Laboratories, Grand Island, NY) supplemented with 20% heat inactivated fetal bovine serum (FCS, catalog # 210—6510, GIBCO Laboratories, Grand Island, NY) 100 ug per ml Gentamicin‘E (Schering Corp., Kenilworth, NJ) and 1 unit per ml heparin. The culture medium was further supplemented with combinations of 0, 0.25, 0.5 or 1.05 mM pyruvate or 0, 25 and 50 mM lactate. These levels were based on work of Brinster (1965). The glucose—free 199 medium was specially prepared by GIBCO Laboratories (catalog # 82-0060, Grand Island, NY) and used in the second series of pyruvate—lactate in vitro fertilization trials. The concentration of glucose in fetal calf serum was between 133 to 277 mg per decaliter. All media were sterilized by filtration through a 0.45 pm Millex filter (Millipore Corp., Bedford, MA) and stored in 10 ml vacutainer tubes (2°C). Fresh culture medium was prepared every 3 weeks. The concentration of hypotaurine (Sigma Chemical Company, St. Louis, Missouri) tested in the in vitro fertilization system was 0.5 mM. The motility of squirrel monkey sperm cells was also assessed in 0.5 mM taurine (Sigma Chemical Company, St. Louis, Missouri), 0.5 mM hypotaurine and 70 pM L—epinephrine (Sigma Chemical Company, St. Louis, Missouri), in the following experimental design: (1) control, (2) hypotaurine, (3) taurine, (4) hypotaurine and taurine, (5) epinephrine, (6) hypotaurine and epinephrine, (7) taurine and epinephrine, (8) hypotaurine, taurine and epinephrine. The 32 levels tested were selected based on reports of Bavister gt gt., (1979) and Meizel gt gt., (1980). Human cord serum (HCS) was collected from patients at St. Lawrence Hospital, Lansing, Michigan. HCS was centrifuged and the supernatant stored frozen. Hemolysed HCS, distinguished by red coloration, was not used. The HCS was heated at 560C for 30 mins, filtered through a 0.45 pm Millex filter and added to medium 199 at a level of 20 percent. Criteria of maturation and fertilization At intervals of 24 hours, the oocyte cultures were examined through an inverted microscope and the stage of development noted. The presence of a polar body on an oocyte indicated a mature oocyte at metaphase II. The criteria for fertilization were the presence of: (1) two or more polar bodies in the perivitelline space (2) two or more polar bodies and two pronuclei or two or more equal sized blastomeres by 24 hr after insemination (3) verification of two or more sets of chromosomes through Giemsa staining (Mizoguchi and Dukelow, 1980) (4) observation of the sperm tail or midpiece within the cytoplasm. When an oocyte fulfilled one or more of the above criteria, it was designated as fertilized. An oocyte was considered dege— nerated when either the oocyte had become black oocyte had shrunken into an uneven shape. 33 Tripleestaining of squirrel monkey sperm cells The purpose of the triple—stain (Talbot and Chacon, 1981) is to determine if the sperm cells have undergone the acrosome reaction, a necessary step prior to sperm penetration of the oocytes. The triple—stain procedure involved incubating the sperm cells in 2% Trypan Blue stain (a stain which distinguishes live from dead sperm cells) at 370C for 15 minutes, washing in culture medium and centrifuging at 1800 rpm for 10 minutes in a Sorvall RC2—B centrifuge. The pellet was fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hour, centrifuged and the pellet washed and centrifuged twice. The pellet was air—dried, and 0.8% Bismark Brown Y (post—acrosomal contrast stain, pH 1.8, 400C, 5 mins) used to stain the sperm cells. After washing, the third stain, 0.8% Rose Bengal (acrosomal region stain, pH 5.3, 24°C, 45 mins) was used and the washed sperm cells were then air—dried and observations made under a light microscope. Live acrosome—reacted sperm cells had light brown post—acrosomal regions with a white acrosomal cap (Figure 4). Dead sperm cells had dark blue—black post—acrosomal regions. Pink acrosomal caps indicated that the acrosomes were intact. Statistical analysis of data Dunnett's t—test for pairwise comparison with the control was used to analyse sperm cell motility. The percent oocyte PINK WHITE PINK DARK BLUE BLACK 1.Dead sperm 2.Dead sperm 3.Live sperm 4.Live sperm cell with an cell with cell with cell with intact reacted intact reacted acrosome acrosome acrosome acrosome Figure 4. Four staining patterns observed after staining sperm cells with the triple stain. ( albot and Chacon, 1981 . 35 maturation and fertilization were compared using 2x2 contingency chi—square analysis. If the trials had more than one treatment, then Bonferroni chi—square test for multiple comparisons was used. For treatment groups with less than 10 oocytes, a more stringent Fisher's exact test was used. All analyses were computed using the Hewlett Packard 41C statis— tical packet. The ANOVA tests for the lactate, pyruvate and glucose studies are presented in Appendix I. Experimental design Throughout the studies, all possible steps were taken to reduce biological variation. Oocytes in all groups were randomly selected from the pool of oocytes recovered by lapa— roscopy. One to three oocytes were assigned to each well in the culture slide. Sperm cell motility studies were done such that in each trial, the sperm cells in the control and treated groups were from the same male. The treatments in each study were randomly assigned to the sperm cells. Each culture containing oocytes received the same approximate concentration of sperm (105 to 106 sperm/ml). The triple—stain procedure (described on page 33) was utilized to test the hypothesis that dbcAMP accelerated the acrosome reaction in sperm cells. The squirrel monkey sperm cells were divided into two groups; one group was treated with 1 uM dbcAMP and the other group served as a control. Aliquots 36 of sperm cells were removed with a pasteur pipet from the two groups over a 3-hour period and stained using the triple—stain procedure. The percentage of sperm cells staining positive for acrosome reactions was recorded. The chi—square statistic was used because of the quantal nature of the in vitro fertilization data. The Dunnett's t-test was chosen to evaluate sperm cell motility because it allowed comparison of the data from each individual treatment with the control treatment. On various occasions, standard ANOVA tests were also utilized, as indicated in the text, and with tabular summaries in the Appendix. In presenting the data, all significant differences were specifically noted. If there was no specific notation, the groups (or cells) in the tables show no statistically signifi— cantly differences. The failure to achieve statistical signi— ficance may be attributed to a variety of possible (but un— tested) reasons. However, the particular design was chosen in each case to include at least a partial replication of earlier work (often in another species) where the author claimed sta— tistically significant differences for certain treatments compared to their controls. RESULTS IN VITRO MATURATION OF SQUIRREL MONKEY OOCYTES I. Effect of pyruvate and lactate supplementation on oocyte maturation in a glucoseecontaining medium. The result of testing combinations of 0, 25 and 50 mM lactate and O, 0.25, 0.5 and 1.05 mM pyruvate in glucose— containing medium 199 are shown in Table 1. No significant interaction between pyruvate and lactate was found for the maturation of squirrel monkey oocytes. Only the 50 mM level of lactate treatment resulted in a significantly higher percent of oocyte maturation compared with the 25 mM lactate group (p < 0.05) when tested by the Bonferron procedure. There were no differences in oocyte maturation in the 0, 0.25, 0.5 and 1.05 mM pyruvate treatment groups. The overall percent of oocyte maturation was 36.6%. These data failed to confirm the direct applicability of the findings of Brinster (1965) in the mouse, despite the design of the experiment to provide two way dose—response data which might have revealed interactions between pyruvate and lactate, if present. These points are discussed later. II. Pyruvate and lactate supplementation effects on oocyte maturation in a glucoseefree medium. The percent maturation of squirrel monkey oocytes 37 38 EOHM HOG. “DD Iii “mumuoma mo Hm>ma 25 mm m£u Eonm ucmhmmmwp %Hucmofiwwcwwmm .Amo.o V my Hopucoo mfiu no.0mcmom\aaa Am.wmcwma\aq im.qmcmo\qN AN.omch\om im.chmm\mH Hmoon was.chom\aq in.wqcnm\ma La.mmcmm\m no.mmvaa\m Am.mqVMN\on om Aw.annm\NN no.omvom\nfi Lo.wmcmw\n Am.oNcmH\m Am.mmvaa\s mm Aa.nmceaa\mq im.mmcmo\mm AH.mmcHN\m Lo.oscma\o in.omvma\q o - -- - - Axes Hmoon mo.a om.o mN.o o ,coHomuocmocoo mumuomq AZEV aowumuuamocoo mum>thm ...... ARV mmumooo pmho>oomm mo .oz\pmunumz mo .02 l . . . .AH xwpfimda< mom n<>oz< Homv EDvaE wcflcwmucoolmmooSHw CH mmumooo %MXGOE Hmphfinvm mo cowumhdume OhuH> CH ozu so oumuoma paw oum>flu>m mo uommmm .H mHan 39 cultured in 0, 25 and 50 mM lactate and (or) 0, 0.5 and 1.05 mM pyruvate in glucose—free culture medium are shown in Table 2. There were no significant differences for the lactate levels tested. The 1.05 mM pyruvate—containing groups had a lower (p < 0.15) percent maturation (91%) compared with the pyruvate—free groups (22.7%). The overall percent maturation was 14.6 percent, and was significantly lower (p < 0.01) than that of the glucose—containing group (36.6%; Table 1) suggesting that glucose is required for maturation. The design for this experiment was the same as for the previous experiment, except for the omission of glucose from the medium. However, a total of 82 oocytes was used (in contrast to the 303 oocytes reported in table 1). The overall design for the two experiments envisioned a unified 3-dimen— sional statistical test for the data. Due to the small numbers of maturing oocytes per test cell, this possibility had to be abandoned. These results are discussed later. III. Effect of human cord serum on oocyte maturation. Human cord serum contains three growth promoting factors (Hsu, 1980) and have been shown to enhance development of preimplantation mouse embryos in vitro (Chen and Hsu, 1982). Human cord serum was tested for its effect on oocyte maturation. The results (Table 3) indicate that human cord serum (20%) significantly enhanced oocyte maturation (48.6% in HCS compared with 25.0% in the control). 40 .AmH.o v av HOhucOo Scum unmnmmwfip haucwowmwcwwmm Ao.qfivmm\ma mAH.mvmm\m Am.¢fivmm\q Am.NNVNN\m HonouDSm in.oacqm\q AH.mVHH\H Am.qHVN\H Am.mmvo\m on Am.macom\q AH.oVHH\H AH.HHVm\H Lo.omcofi\m . mm Am.qfivwm\q AH.mvHH\H , AN.wHVHH\N Am.oavo\a o - ................................... - ..... ,---- AZEV Hmuoonsm mo.a om.o o cohomuoawocoo AEEV cowumhucmoaoo mum>ahhm unwound .AH anamaa< mom n<>oz< Homv fiancee commumwousaw CH mmu%ooo hmxcoe Hohhwsvm Mo cowumudume onuw> CH can So cowooma paw oum>5u>a mo uommmm osH .N mHan 41 .mCEDHoo o>HmmoooDw comaumn oocopcmmwu ou mop mcfiHomp mam>flmmmhwonm m I q mGEDHooa .mma EDHUoE mnnuaso memHH u omHoH .Esnmm mamo Hmumm n mom .Amo.o v av HOMDGoo Eoum uCoHMMMHp maucmoHMflcmem Ao.ov no.va AH.an «Ho.qu w\o wxm mH\w mM\mH n ma AmmHoHfiv Ednom phoo amen: Am.qav Ao.wNV Ao.omv Ao.mmv m\H N\N qH\m om\qa 0 am Amom w mmHoHv wHouucoo ANV mouzooo ANV “NV ANV mHmHHu meEHcm ucwEumeH pmNHHHunmm mmu%ooo mmu%ooo mmuhooo mo .02 mo .02 \mouhooo UoNHHHuHmm pohDumE Hmuou HHoo know \mwumooo \mouhooo \wouhooo Ho conga Haoolosu UmNHHHunw Consume mo .02 mo .02 mo .02 mo .OZ< .cowuwNMHHuuom Ucm coaumhsume ouzooo so EDme UHoo amass sums coaumuamamaaadm Mo wuoowmm .m magma 42 IV. Effect of hypotaurine on oocyte maturation. Hypotaurine, a beta-amino acid, is found in hamster follicular fluid and macaque oviductal fluid (Meizel gt gt., 1980) and it enhances sperm motility. When 0.5 mM hypotaurine (a level selected based on work done by Meizel gt gt., 1980 on hamster sperm cells) was added to the basic culture medium, (Table 4) it had no significant effect on squirrel monkey oocyte maturation (38.7% in the hypotaurine group compared with 34.1% in the control). V. Effect of time of HCG administration on oocyte maturation. The result of the study comparing two protocols of hormone administration (HCG given 10 hours after or concurrent with, the last FSH injection) is presented in Table 5. There was no difference between the treatments on oocyte maturation. 43 Am.qov HM\ON Am.oav o\H Ao.omv NH\© Am.wmv fim\NH mfiHHDmuomhm SE m.o Am.mov ¢<\mm Ao.mmv m\w Am.mmv ma\w AH.qu ¢¢\ma HOHucou “NV mmuhooo ARV mmuhooo ANV mmumooo ANV mmu%ooo uaoEummnH Hmuou\mhsos wq pmNHHHuHom Hmuou\wmu%ooo Hmuou\mmu%ooo kn poumhmcowmu \mo%HnEm UmNHHHuHmm pmhnume mo .02 mmuxooo mo .oz Hamolm mo .02 mo .02 cowumnwafluumw paw coaumwdume muhooo co mcfihsmuomhn SEm.o mo uomwwm .q canoe 44 AH.qu Am.mwv An.mfiv Amvfi mH\w «O\oa mmc\ooa Nm OH we: nuHB Deohhso Icoo co>fiw 3mm ummq Am.mmv AH.qMV Am.nav ANVH mH\w qq\ma mm¢\ow am 0 com %n nouma H: OH poBOHHom mmm ummq Awwmum ARV ARV ANV mhmxcoe mamflku cowumuuchHEUm Haoo Hmcwmv mouhooo moukooo woumhwamm mo mo mo m0thEo pthumE Hmuou moHoHHHow .oz .oz mth anoHo>op \mmu%ooo \mouhooo wo .oc we .02 ponwamunwm pounume \mmu%ooo mo .02 mo .02 mo .02 Go moon 3mm Hmcfiw «nu Eouw manpowmamm Ho HmauowOD Umuummcfl 00: mo uoommo onfi .coHumNHHHuHom paw goaumunume ouhooo OHUH> CH zoxcoe Hohhfinvm. ..m «Home 45 VARIABLES INVOLVED IN SQUIRREL MONKEY SPERM CELL MOTILITY I. Effect of pyruvate and lactate on sperm cell motility. Addition of 1.05 mM pyruvate significantly (p < 0.05) elevated the percentage of motile sperm cells compared with controls (Figure 5). Addition of 50 mM sodium lactate did not enhance motility above control values and quenched the stimulatory action of pyruvate. There was no difference in sperm cell motility between the saline and the phosphate buffered saline (with pyruvate) groups. Culture medium TC199 supplemented with pyruvate (the usual medium for in vitro fertilization controls) enhanced the motility for only 1 hour and'did not present any exceptional stimulatory action suggesting that the glucose component in medium TC199 did not play a role in sperm motility action. II. Effect of human cord serum on sperm cell motility. Sperm cell motility was enhanced in culture medium supplemented with human cord serum when compared with culture medium supplemented with fetal calf serum (Figure 6). The motility—stimulating effect of cord serum was observed in cultures kept at 200C (room temperature) and at 3700 (p < 0.01). The motility was higher at the 20°C temperature in both serum treatments (p < 0.01). By 22 hours, the motility of both serum treatment groups at 37°C was close to zero while 401 N c? PERCENT MOTILITY Figure 5, 46 30 min ‘fiifi 2hr 7': 1hr .:I:1 3hr lira. P 199 com SAL PBS SAL PBS SAL PBS 199 COMB SAL PBS SAL BS SAL W33 LAC LAC PYR PYR PYR Energy substrate supplementation effects on squirrel monkey sperm motility (SAL-saline; PBS-phosphate buffered saline; 199—medium 199; LAC—50 mM lactate; PYR—1.05 mM pyruvate; COMB-TC199, lactate & pyruvate). *Significantly different from saline and PBS controls (p§;EOMOS). Bar graph values: mean (of 5 observations) + . . . 47 .z.m.m H AmCOHum>nmmno m mow some mH ucHOQ sump Loom .mHHmo Ehomw hmxcoe Hmnhwdvm mo huflafluofi ecu Go Enhom wHwo Hmuwm paw enuom UHoo amen: mo muomwmm HomucmmeMHQ .o mudwflm So: u m V M O . .l . . N . I 0 ‘ll‘wl I I, Altwliazir . / O ’ O ‘ ' . ’ O O O O /4:£yr. ’ I o R meal. . O Y*§§ttwagori v / v ¢;::i¢. I o. .Wittnttt t I Well-3000 .C‘ A: I 0'01!!! ‘ "-::. . , . . oonm mo: .’ z . a . . . WIN N o o O O. o Jad‘q b 6' )USOJad é It (I: ooow mow/v... ..... w ‘ T . . I. . n It" 0 l" O I Oil " . c I 0 fl ’ V ¥ ’ b . KINIJOW / / owl ooo~ mom LY/ 7.. a, a 48 sperm in treatment groups at 20°C were 25 percent motile. HCS clearly sustained sperm cell motility. III. Effect of hypotaurine on sperm cell motility. The percentage of motile sperm cells was significantly higher (p < 0.05) in the hypotaurine (0.5mM) treatment group (Figure 7) when compared with the control. This effect was maintained for up to 4 hours. Taurine (0.5 mM), a related beta—amino acid, did not stimulate sperm cell motility. Epinephrine (70 pM), a hypothetical co—factor of hypotaurine (Meizel and Working, 1980) involved in stimulating hamster sperm acrosome reactions, did not enhance squirrel monkey sperm cell motility either alone, or in combination with taurine and hypotaurine. The motility—enhancing action of hypotaurine was quenched by combining it with the other compounds. After 5 hours of incubation, all treatment groups exhibited the same low level of motility (14.9 i 1.7 percent motile). IV. Effect of calcium on sperm cell motility. Calcium ions promote guinea pig sperm cell acrosome reaction (Yanagimachi and Usui, 1974) and enhance ciliary motility (Blum gt gt., 1980). In contrast, Tash and Means (1982) showed that calcium ions inhibit dog sperm cell motility. The question of whether or not calcium ions affect squirrel monkey sperm cell motility was investigated. The 40* Figure 7. 49 * TIT :3()TTHIT :3t1r I m H T * I 7 ' 7 T7 1hr 4hr ;:]:;AI] [1; 1-‘r * I} 2hr 5hr l]; ::[:j::[:I:];j 1f.r L...- C HT T HT E HTT C HT T HT E HT T HT TIE T E E TIE The effect of hypotaurine, taurine and epinephrine on squirrel monkey sperm motility. (C—control; HT—0.5 mM hypotaurine; T-0.5mM taurine; E-70 pM epinephrine). *Significantly different from control (p < 0.05). Bar graph point : mean (of 5 observations ) i S.E.M. 50 levels chosen were based on published work in other species (Yanagimachi and Usui, 1974; Hyne and Garbers, 1979; Parrish- and Polakoski, 1981). The results of the study were as follows: the percent motile sperm cells after 30 minutes of incubation (Figure 8) was initially lower in 3 treatments (1.2, 2.3 and 4.2 mM calcium) compared with the control. The motility in the control group then declined with time while the 7.1 mM calcium—treated group maintained a significantly higher motility over a period of 5 hours. Only two other levels of calcium treatment showed enhanced motility; the 1.2 mM calcium group at hour 2 and the 2.3 mM calcium group at hour 3. V. Effect of follicular fluid on sperm cell motility. Follicular fluid contains an dialysable factor which promotes hamster sperm cell motility (Yanagimachi and Chang, 1964; Barros and Austin, 1967). The follicular fluid used in the present study was aspirated from the ovarian follicles of anesthetized squirrel monkeys. The object of the research was to determine if squirrel monkey follicular fluid would support sperm cell motility in a manner similar to that observed with hamster sperm cells. The results (Figure 9) showed that follicular fluid enhanced sperm cell motility for up to 23 hours. Filtration of the follicular fluid through a 0.45 pm millipore filter decreased motility enhancement suggesting the involvement of 51 .z.m.m H Amcoflum>uomno m wow some mm u¢fl0d wouuoad.nomm .Amo.o V.ac morocco sown oompmmmno snocmonmncwnmr .muwaflu06 Haoo Ehmam %mxcoe HoHHHDUm Co EDHQHmo mo uommwo age .w wHDth Kaltlnow 3U9319d IOOH .z.m.m H AmCOHumSHomno-m mov Con mm ucflom pmuuoad comm .nmo.o v av HonuCoo Eonm ucohomwfiw haucmofiwflcwflmx .muHHHuoE HHmo EHon hmxcoe Houhwdvm co UHDHM HmHDoHHHow mo uoommm oLH . m ohnwflm Hsom mm m q m N H o c x x _ p L . a . O N :ON UHDHM Hmaaoflaaom poHouHHm - d m too a 2 a 5 3:: Hansonfion m. 4 N o 1 It Too H .J. ,A low SS 53 large macromolecules in enhancing squirrel monkey sperm cell motility. The nature of follicular fluid and its ability to. enhance sperm cell motility is discussed in the next section. VI. Effect of dbcAMP on the sperm acrosome reaction. The triple stain procedure allows one to distinguish acrosome—reacted sperm cells (and hence, ready for oocyte penetration and fertilization) from unreacted sperm cells. The purpose of the experiment was to determine the effect of 1 uM dbcAMP on the acrosome reaction (the triple stain was used as an assay for the acrosome reaction). The results (Figure 10) showed that dbcAMP accelerated the'acrosome reaction in the sperm cells. After the first hour of incubation, the dbcAMP—treated group showed a signi- ficantly higher (p < 0.05) percent sperm cells with the acrosome reaction (32.5 i S.E. 5.6) when compared with the control group (10.0 i S.E. 0.0). Similarly, at hour 2 of incubation, the dbcAMP group had more acrosome-reacted sperm cells than the controls. There were no differences in the treatment and control groups after 3 hours of incubation. .z.m.m H chomum>homno m mov cmoE ma Damon panhw Hop 30mm .Amo.o v av Honucoo EOHM uConMMHU haucmowmwcwflmr . mHHoo Summm >mxcoE HoHHHDUm mo Gowuuwoh oEowOHom onu so mz/ I o A... Myrna/m3.” M, 4 /. . b ./w/¢ a o o o ,flv..,n,¢o~¢oo’o 54 / she. 5.. //¢/ , .fl . //.//moonoOOOoo / 11 O O 0 b x // 0.000009. / I O O Q . . fl/ 0000.050. // HW/ .3900... t, r , Q o MMu/n/ .0. to. o {nu/.IZ/ 0.0000000 / ///, O I ”.1. a 0.00... l . O . C O D O O ‘ O C O I 0 00.090000 6 A a O t asshmm mumuomq .EDHUmE wcwcwmuCoolwwooDHw EH mmumooo hoxcoe Hohhwsvm Mo CoHumNflHfluhmw Go oumuoma paw oum>5nxa mo Doomwm .o oHAMH 57 .Aoa.o.vav Ho>oH oumuoma 25 mm pan 0 Eoum unmhwwmwp haucmofimmcwflmm in.HqVNH\m Am.mmcm\a Ao.omcq\w Lo.oqcm\m neoconsm mio.ooacq\q Ao.ooaca\a Lo.ooaca\a no.00HcN\N on Ao.ovq\o Lo.oca\o Lo.oca\o no.ocm\o mN Lo.mmcq\fi Lo.ocfi\o . Ao.och\H Ao.oca\o o a..(... I...ut.\o..-......\(.-..(v.-l;t.......t.u(l(1:ol-c-Tvltl....c..r: AZEV Hmooonnm mo.H om.o o conomnocooooo ASEV aowumHDCmucoo ouw>thm mumuomq .EDHme mmnmnmwoosaw CH mmuhooo hoxcoe Hohhfisvm mo :oHuwNHHHuHom onuw> SH can no mumuoma paw mum>5H%Q mo uoommm mnH .m.mHan 58 III. Effect of human cord serum on in vitro fertilization. The results (Table 3; page 41) indicated that human cord serum had no effect on fertilization (8/17; 47.1%) compared with the control (7/14; 50.0%). IV. Effect of hypotaurine on in vitro fertilization. There was no difference in the percent fertilization in cultures (Table 4; page 43) treated with 0.5 mM hypotaurine (6/12; 50.0%) and the control (8/15; 53.3%). V. Effect of time of HCG administration on in vitro fertilization of matured oocytes. In the first protocol (final FSH and HCG injections given 10 hours apart), 8 oocytes were fertilized out of 15 matured oocytes (53.3%; Table 5, page 44). The second protocol (final FSH and HCG injections given concurrently) resulted in 8 fertilized oocytes out of 19 matured oocytes. The percent in vitro fertilization was not different for the two groups. 59 CLEAVAGE OF IN VITRO FERTILIZED SQUIRREL MONKEY OOCYTES I. Effect of pyruvate and lactate on first cleavage in glucose—containing medium. The number of embryos continuing in culture (Table 8, page 60) were limited and no statistically significant differ— ences were found in the treatment groups (treatment combina— tions of 0, 25 and 50 mM lactate and 0, 0.5 and 1.05 mM pyruvate). The overall percent cleavage was 17.5 (10 two—cell embryos from 57 fertilized oocytes). II. Effect of pyruvate and lactate on first cleavage in glucoseefree medium. Five in vitro fertilized oocytes were cultured but no cleavages were observed. The number of oocytes tested were limited due to low oocyte maturation (12/82; 14.6%; Table 2, page 39) in glucose—free culture-medium. III. The effect of human cord serum on first cleavage of fertilized squirrel monkey oocytes. There was no difference in the percent cleavage in the 20% human cord serum treatment (2/8, 25.0%; Table 3, page 41) compared with the 20% fetal calf serum control treatment (2/7, 28.6%). One of the two—cell embryos in the control treatment developed to a four—cell embryo. Am.mfivnm\oH Ao.NNVHM\m Am.wVNH\H Ho.ovm\o Ao.oqvm\m HNDOH Aw.MHVNN\m Ao.mmvm\m Am.ofivo\fi Ao.ovm\o Ao.oVM\o om Ac.wquH\q Ao.oqvm\m Ao.oVM\o Ao.ovq\o Ao.ooHvN\N mm AN.¢HVHN\m An.oavwfi\m Ao.oVM\o II II o w - , - , - .- - - AZEV amuOH mo.H om.o mN.o o cofiDMHucmoGou AZEV cowumHDCwocoo mum>shhm wumuomq EL 86:38 BNEBSE mo .oziofinem :8 N no .02 .EDHme wcficflmucoolmmoonaw CH mmukooo hmxcoa Hohhwdww mo wwm>mon so mumuomH paw mum>3phm mo uoowmm .m mHan 61 IV. The effect of 0.5 mM hypotaurine on first cleavage of in vitro fertilized squirrel monkey oocytes. Two out of the 8 in vitro fertilized oocytes (25.0%) in the control group cleaved to the two—cell stage (Table 4, page 43). The 0.5 mM hypotaurine—treated group had one 2-cell embryo (out of 6 fertilized oocytes; 16.7%). There was no difference between the 0.5 mM hypotaurine—treated group and the control. DISCUSSION Oocyte.maturation A study of the interactions of lactate (0, 25 and 50 mM), pyruvate (0, 0.25, 0.5 and 1.05 mM) and glucose (5.6 mM) showed that glucose was required for the maturation of squirrel monkey oocytes. This was reflected in the higher percentage of matured oocytes in cultures containing glucose compared with glucose—free cultures. This is a significant finding because there has been no research on the effect of energy substrate on immature primate oocytes. Lactate, which did not show any effect by itself, synergized with glucose'(or facilitated glucose utilization) to increase maturation. In the absence of glucose, pyruvate, but not lactate, was inhibitory on maturation. Fridhandler (1961) showed that rabbit oocytes metabolize energy substrates only through the pentose phosphate pathway. If this is also true with squirrel monkey oocytes, then glu- cose but not the other two substrates should promote matur— ation. The results of the study show such an effect, but further demonstrates that lactate and glucose have a enhancing effect on oocyte maturation. Two other variables that are important are the pH of the medium and the transport carrier molecules on the oocyte 62 63 plasma membrane. The pH affects the charges on the substrate molecules. If the substrate becomes negatively charged, it may go against an electrical gradient in passing into the oocyte (the interior of the oocyte is negatively charged). Carrier molecule (facilitated diffusion) transport at the oocyte plasma membrane is dependent on the concentration of substrates in the medium. High external concentrations of substrate facilitate substrate entry into the cell. Different substrates may compete for the same carrier and reduce the efficiency of substrate transport into the cell (Hendrix, 1980). It was hypothesized that the substitution of human cord 'serum for fetal calf serum would provide the three differen— tiation factors (EGDF1—3) (Hsu, 1980) for the oocytes and promote development. A significantly higher maturation rate was seen with cord serum in the present studies. The use of human cord serum results in an approximate doubling of the percentage of matured oocytes in the culture system and this is important both for research production of in vitro fertil— ized embryos and in human embryo production (because of the difficult nature in collecting matured primate oocytes). The explanation for the higher percent oocyte maturation seen in the cord serum treatment (compared with fetal calf serum) is unknown. It is not an effect of steroids in the cord serum since steroids (progesterone, estradiol, andro— stenedione) inhibit oocyte maturation (Robertson and Baker, 64 1969; Nekola and Smith, 1974; McGaughey, 1977; Eppig and Koide, 1978; Smith and Tenney, 1980). It is possible that the 3 unidentified growth factors (Hsu, 1980) in the cord serum promote oocyte maturation but further studies must be done to identify the exact stimulatory mechanism. The present studies show hypotaurine (0.5 mM) has no effect on oocyte maturation. This beta amino acid found in follicular fluid (0.46 — 0.47 mM; Meizel gt gt., 1980) probably does not play a physiological role in the process of oocyte maturation. Its function appears to be the activation of sperm cells (discussed in the next section). Motility of sperm cells Squirrel monkey sperm cells have different energy source requirements than oocytes. The studies on sperm cell motility emphasize the stimulatory effect of pyruvate (1.05 mM) on motility in the absence of the other energy substrates. When both lactate (50 mM) and pyruvate were tested together, the motility of the sperm cells was not different from the control. One possible explanation for this observation is that the addition of both pyruvate and lactate causes an imbalance in the redox potential resulting in inefficient utilization of substrates. An alternate explanation is that these substrates compete for the same carrier molecule. Quinn and Wales (1973) observed that the transport of pyruvate (as measured by labelled pyruvate) through cellular membrane can 65 be inhibited by lactate. In such a case, lactate blocks sperm metabolism by inhibiting uptake of pyruvate into the sperm cells, resulting in low motility.' The studies on the effect of human cord serum (at a level of 20% in culture medium) on the motility of sperm cells clearly demonstrated that this serum enhanced motility when compared with the fetal calf serum treatment. It is post— ulated that motility factors, similar or identical to the factors found in blood serum (Yanagimachi and Chang, 1964; Lui gt gt., 1977; Meizel gt gt., 1980) are responsible for stimu— lating motility. The studies on hypotaurine (0.5 mM) showed that this compound stimulated the motility of squirrel monkey sperm cells. This was consistent with observations in the hamster (Meizel gt gt., 1980). However, the observations on the effect of taurine and epinephrine on motility were in contrast with observations made in the hamster (Cornett and Meizel, 1978; Meizel and Working, 1980). In those studies the motility of hamster sperm cells were stimulated by the same levels of taurine and epinephrine used in the present studies. Squirrel monkey sperm cells were not stimulated by taurine and epinephrine. This suggests that squirrel monkey sperm cells either lack the receptors for taurine and epinephrine or that the levels tested were too low. Based on the observations in this study, the physio— logical function of hypotaurine (found in the female repro— 66 ductive tract) is to stimulate motility of squirrel monkey sperm cells. The trials involving sperm incubation in various calcium concentrations showed high sperm motility in the 7.1 mM calcium group. There was no difference in the motility of sperm cells incubated with 1.2, 2.3, and 4.2 mM calcium. The observation that calcium affects the sperm cell has been demonstrated for other species (Yanagimachi and Usui, 1974; Reyes gt gt., 1978). However, the reports are conflicting since calcium stimulated motility in some species but inhi- bited it in others (discussed on page 17). The present studies were carried out to determine the effect of this divalent ion on squirrel monkey sperm cells. The rationale was that the culture medium (TC199) used for in vitro fertil— ization contained calcium (1.0 mM). It was necessary to determine if calcium ions were detrimental to fertilization. The studies showed that calcium ions (at 7.1 mM) enhance the motility of sperm cells. The experiment on preovulatory follicular fluid showed that this fluid maintained the motility of squirrel monkey sperm cells up to 23 hours. Yanagimachi and Chang (1964) showed that factors in bovine follicular fluid were able to induce capacitation in hamster sperm cells. They identified three factors in follicular fluid, a heat—unstable complement, a dialysable component (implicated in enhancing motility of 67 sperm cells), and a non—dialysable component (shown to stimulate sperm cell acrosome reactions). The possibility existed that the factor in follicular fluid responsible for enhanced motility was hypotaurine. This is plausible for two reasons: (1) hypotaurine has been identified in follicular fluid (Meizel gt gt., 1980) and (2) hypotaurine has been shown to stimulate the motility of squirrel monkey sperm cells (page 49). There are two arguments against this reasoning: (1) the motility of sperm cells in follicular fluid was sustained up to 23 hours whereas hypotaurine enhanced motility for only 4 hours in the present studies, and (2) filtered follicular fluid did not enhance motility suggesting the involvement of large macromolecular factors, rather than a small molecular weight compound like hypotaurine (molecular weight 109.1). Earlier studies demonstrated that dbcAMP treatment increased the percentage of in vitro fertilized oocyte when compared with the control (Chan gt gt., 1982). It was thus hypothesized that the action of dbcAMP was to enhance the acrosome reaction. The present study tested this hypothesis. A triple—stain technique was used to distinguish acrosome— reacted from unreacted sperm cells (Talbot and Chacon, 1981a). The results showed that dbcAMP (1 pM) accelerated the sperm cell acrosome reaction when compared with untreated sperm cells. This suggests that under natural conditions, cyclic AMP (either from the cumulus cells or cells lining the 68 reproductive tract) is involved in sperm activation. Sperm cells coming in contact with cyclic AMP undergo the acrosome reaction and consequently fertilize the oocytes. In vitro fertilization The present studies demonstrated that the addition of pyruvate (1.05 mM) to the culture medium increased the percentage of fertilized oocytes above the control (0 mM pyruvate) in the presence of glucose (5.6 mM). This effect is probably through the effect on the sperm cells since it was previously shown (page 45) that pyruvate enhanced the motility of sperm cells. One could argue against this action of pyruvate on the sperm cells because the motility of sperm cells in culture medium with 1.05 mM pyruvate was higher than with medium containing pyruvate and glucose (Figure 5). One would expect to see a higher percent fertilization in glucose— free medium containing pyruvate, than in medium containing both pyruvate and glucose. The data showed that this was not true (Table 6 and 7), suggesting that the effect of the combi— nation of energy substrates was on the oocyte rather than on the sperm cell. The studies also demonstrated that the addition of glucose to the cultures in the absence of other substrates, had no effect on fertilization. This suggests that at fertil— ization, glucose facilitates transport of pyruvate into the sperm cells or oocytes. This is suppported by the fact that 69 in the absence of glucose, pyruvate had no effect on fertil- ization (Table 7). The present studies also showed that lactate (10 mM) enhanced fertilization in the absence of glucose. This suggests that glucose or its metabolites inhibit the uptake and metabolism of lactate in the oocyte. It is postulated that at fertilization, glucose acts as a regulator in the oocyte, promoting the utilization of pyruvate while inhibiting lactate utilization. In the absence of the regulator (glucose), lactate is utilized in preference to pyruvate. Human cord serum enhanced the motility of sperm cells when compared with fetal calf serum. However, cord serum did not affect fertilization. Apparently, the factors in human cord serum only play a role in sperm cell motility and oocyte maturation (shown in the present studies) and at embryo development (Hsu, 1980). The present results indicated that hypotaurine (0.5 mM) had no effect on in vitro fertilization. This compound interacts with beta-adrenergic receptors (Wheler gt gt., 1979) and activates adenylate cyclase to produce cyclic AMP. Cyclic AMP has been implicated in microtubule disassembly in cell division processes. The squirrel monkey oocytes either do not have beta-adrenergic receptors or the cyclic AMP produced was rapidly metabolized to inert S'AMP by phosphodiesterase. 70 Cleavage of fertilized oocytes Results of the present studies support the hypothesis, that glucose is required for embryo cleavage. In the absence of glucose, no cleavage occurred. This is consistent with the observations of Fridhandler (1961) on rabbit oocytes. Results of the present studies also showed that pyruvate and lactate had no effect on the first cleavage of squirrel monkey embryos. These observations were limited as the number of available embryos for study was small. Brinster (1965) has, however, demonstrated interactions of energy substrates on the in vitro development of in vivo fertilized mouse embryos and found that the optimum concen— trations for lactate and pyruvate were 50 mM and 0.5 mM respectively. Tissues appear to have set equilibrium levels of lactate, pyruvate and other energy sources at a particular cell cycle stage, and unless substrates are added in concen— trations that are balanced, then the metabolism machinery is interrupted (Brinster, 1965). Observations on the cleavage of fertilized squirrel monkey oocytes treated with human cord serum (20%) or hypo— taurine (0.5 mM) were also limited. There was no difference in first cleavage with these treatments compared with the control. SUMMARY AND CONCLUSIONS The squirrel monkey in vitro fertilization system is a useful non-human primate system for the study of primate reproductive physiology. The studies conducted produced the following results: (1) Oocyte maturation was higher in cultures containing 5.6 mM glucose than in cultures without glucose. Lactate (50 mM) enhanced oocyte maturation in the presence of glucose (5.6 mM). Human cord serum (20%) but not hypotaurine ' (0.5mM) treatment enhanced oocyte maturation. (2) Squirrel monkey sperm cells showed enhanced motility in culture media containing pyruvate (1.05 mM), human cord serum (20%), hypotaurine (0.5 mM), follicular fluid and calcium (7.1 mM), when compared with the control. Lactate (25 mM), taurine (0.5 mM), epinephrine (70 uM) and calcium levels at 1.2, 2.3 and 4.2 mM had no effect on sperm cell motility. (3) Glucose supplementation (5.6 mM) did not affect fertili— zation of oocytes in vitro. Pyruvate (1.05 mM) enhanced percent fertilization above the control in the presence of glucose. In the absence of glucose, lactate (50 mM) enhanced fertilization but not pyruvate. Human cord serum (20%) and hypotaurine (0.5 mM) did not affect the 71 72 percentage of fertilized oocytes. (4) Fertilized oocytes did not cleave in the absence of glucose (5.6 mM). Lactate (25 and 50 mM), pyruvate (0.25, 0.5 and 1.05 mM), human cord serum (20%) and hypotaurine (0.5 mM) treatment had no effect on cleavage of fertilized oocytes compared with the control. In conclusion, the present studies suggested that glucose and lactate played a physiological role in the maturation process of squirrel monkey oocytes. Factor(s) in human cord serum also enhanced oocyte maturation. The studies also suggested that naturally—occurring substances in the female reproductive tract (pyruvate, hypotaurine, follicular fluid and calcium) played a role in sperm motility. The process of fertilization apparently required the presence of glucose and pyruvate. Finally, the studies suggested a role of glucose in the process of first cleavage. 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Hafez, ed.), Mosby, St. Louis, pp. 570-582. APPENDIX I ANALYSIS OF VARIANCE (TWO WAY) APPENDIX I ANALYSIS OF VARIANCE (TWO WAY) Table 9. in a glucose—containing medium. Effect of pyruvate and lactate on oocyte maturation Sum of Degrees of squares, (ss) freedom, (df) F ratio (Lactate levels) row 708.10 2 20.11* (Pyruvate levels) column 139.90 3 2.65 Error 105.63 6 Total 953.62 7ELactate effect is significant (p < 0.05) Table 10. Effect of pyruvate and lactate on oocyte maturation in a glucose—free medium. ss df F ratio (Lactate levels) row 49.78 2 0.77 (Pyruvate levels) column 309.55 2 4.76 Error 129.96 4 Total 489.29 98 Table 11. 99 Effect of pyruvate and lactate on in vitro fertilization in a glucose—containing medium. . .- - .. § . o. - .. .u. -- .. o- a u— .- ss df F ratio (Lactate levels) row 1894.91 2 1.59 (Pyruvate levels) column 1702.34 3 0.95 Error 3570.84 6 Total 7168.09 Table 12. Effect of pyruvate and lactate on in vitro fertilization in a glucose—free medium. 35' df F ratio (Lactate levels) row 16222.22 2 31.0* (Pyruvate levels) column 555.56 2 1.0 Error 1111.11 4 Total 18888.89 ‘_‘l *Lactate effect is significant (p 4 0.05) 100 Table 13. Effect of pyruvate and lactate on first cleavage in a glucose—containing medium. gs df F ratio (Lactate levels) row 2124.23 2 1.27 (Pyruvate levels) column - 2371.05 3 0.95 Error 5007.29 6 Total 9502.57 APPENDIX II A THREE YEAR OBSERVATION OF SQUIRREL MONKEY OOCYTE MATURATION, FERTILIZATION, CLEAVAGE AND DEGENERATION T.Amo.o.vav Hawk deem onu mo mHouHmDU Hwnuo ecu Eoum ufimhmmmap haufimowwwcwflmr .A.m.m + unmoumdv mmuhooo Hmuou\mu%ooo consume we .oc "namhw Hem , .GOHUMHDUME ORUH> aw ou%ooo hoxcoe HmHHHDUw mo hpsum Hawk mmHSu < .HH mhsmflm mcuhm<=a . vmwm emu.“ . 1 9 . ..n.n.».n...wu.n1u £6... o... 8.00.»... .? Q 9 0:. o p p Y a O a. O A. O Q . 0 fi 4 . ooooeoooatoe on... o 9 o .QQQOOOoomflqooOOoohooooo . ' . . . . . . . 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O 00.009.00.000 1“» 'A.A.....o..o’b.p’g?a..a.,oi.afig.,5.‘ ‘ 4 q. o'o'o’o’o‘o’o o’o‘o‘o'.:’o:¢ 9 0.0 ' A9; ‘wWwvflr “o’o’o‘o’o‘o’o’o'o /*t/ 1 234 QUARTERS A three year study of squirrel monkey oocyte degenerat A .... ‘. . . ‘. - b § 0 Q o y o o o o o o oobo‘ooo 00990009000000 ¢ 0 O 9.0.0.03.5....9€.o.0.0.0.0.9. ' 0.0.9.. .o.o‘b’o.y o e 9' .9 9.0.0.0.0..... .o.o.o.¢.o.o.¢.q o 0.0.. ¢ 0 o o o 9 aa‘. Q