Richard L. Stouffer

In vitro fertilization and embryo transfer in primates

This volume contains the proceedings of a symposium which provides an in-depth analysis of current IVF-ET technology as applied to nonhuman primates. Although IVF-ET is now considered standard treatment for several categories of human infertility, a nonhuman primate model remains highly desirable for the study of reproductive and developmental processes, as well as for the establishment of improved disease models for medical research and the preservation of endangered nonhuman primate species. A better understanding of the primate reproductive system is thus essential to facilitate a global effort to control human population and to aid childless families attempting to overcome their infertility.

Progesterone production by luteal cells isolated from cynomolgus monkeys: Effects of gonadotropin and prolactin during acute incubation and cell culture

Corpus luteum function in cynomolgus monkeys (Macaca fascicularis) during the menstrual cycle and immediately following parturition was evaluated through in vitro studies on progesterone production by dispersed luteal cells in the presence and absence of human chorionic gonadotropin (hCG) or human prolactin (hPRL). Luteal cells isolated between days 17-20 of the menstrual cycle secreted progesterone (P) during short-term incubation (21.6 +/- 1.2 ngP/ml/5 X 10(4) cells/3 hr, X +/- S.E., n = 7) and responded to the addition of 1-100 ng hCG with a significant (p less than 0.05) increase in P secretion. Cells removed the day of delivery secreted large, but variable (27.9-222 ng/ml, n = 4) amounts of P during short-term incubation. Moreover, hCG (100 ng/ml) stimulation of P production by cells at delivery (176 +/- 19% of control) was less than that of cells from the cycle of (336 +/- 65%). The presence of hPRL (2.5-5000 ng/ml) failed to influence P secretion by luteal cells during short-term incubation in the presence or absence of hCG. P production by luteal cells obtained following delivery declined markedly during 8 days of culture in Hams F10 medium: 10% fetal calf serum. Continual exposure to 100 ng/ml of hCG or hPRL failed to influence P secretion through Day 2 of culture. Thereafter hCG progressively enhanced (p less than 0.05) P secretion to 613% of control levels at Day 8 of culture. In contrast, hPRL significantly increased P secretion (163% of control levels, p less than 0.05) between Day 2-4 of culture, but the stimulatory effect diminished thereafter. The data indicate that dispersed luteal cells from the cynomolgus monkey provide a suitable model for in vitro studies on the primate corpus luteum during the menstrual cycle, pregnancy, and the puerperium, including further investigation of the possible roles of gonadotropin and PRL in the regulation of luteal function in primates.

Stimulation of Follicle and Oocyte Development in Macaques for IVF Procedures

In many primate species the interactions between and within components of the hypothalamic-pituitary-ovarian axis ensure the maturation of a single follicle and the timely release of one oocyte capable of fertilization around the middle of the menstrual cycle. Knowledge of the processes involved in growth, selection, maturation, and ovulation of the dominant follicle has increased substantially in recent years, particularly from experimental studies in rhesus monkeys (1, 2). The importance of the pituitary gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), in the folliculo- and gametogenic functions of the ovary have been recognized for over 50 years (3, 4), but recent manipulations to stimulate follicular development have added new information on the processes and events controlled by FSH and LH (5, 6). It is clear that methods that elevate circulating levels of endogenous or exogenous gonadotropins (FSH and LH) override the mechanisms that select a single dominant follicle and stimulate the development of multiple follicles and their enclosed oocytes.

IVF-ET in Old World Monkeys

IVF-ET is an emerging technology in nonhuman primates (NHP) since it is based on relatively limited experience in only a few species. Thus, while successful protocols are available for rhesus and cynomolgus monkeys, consistent or wide-spread application is not yet the order of the day. Undoubtedly, substantial species-dependent differences will be discovered, such as the extended menstrual cycle length recently described by us in Macaca nigra (1). This picture is in marked contrast with the extensive experience base now available in the human.

Duration, Amplitude, and Specificity of the Midcycle Gonadotropin Surge in Nonhuman Primates

The midcycle luteinizing hormone (LH) surge signals key changes within the mature, preovulatory follicle, including resumption of oocyte meiosis, lutein-ization of the follicle wall, ovulation, and early development of the corpus luteum (1). The duration of the LH surge during normal ovarian cycles is considerably longer (48 hours) in women (2) and rhesus monkeys (3) compared with many nonprimate species (rats and rabbits, 4–6 hours; sheep and cattle, 10–16 hours). The concept that surge requirements vary for periovulatory events has emerged from studies in nonprimate species with resumption of oocyte meiosis requiring less LH exposure than that for optimal luteal development (4–6).

Cellular Localization of Estrogen and Progestin Receptors in the Macaque Reproductive System

The past few years have seen a dramatic change in our understanding of the intracellular localization of steroid receptors. Previous lines of evidence had suggested that estrogen and progestin receptors were localized in the cytoplasm in the absence of ligand and that ligand binding to receptor provoked translocation of the steroid-receptor complex to the nucleus. Various techniques, including immunocytochemistry in cryosections, have revealed that most steroid receptors are nuclear proteins whether occupied by steroids or not. In addition, most studies of the hormonal regulation of receptors have been done on extracts of homogenates of whole tissues. Generally this work has indicated that estrogens increase the amount of both the estrogen and progestin receptors and that progestins suppress the level of both receptors. Immunocytochemistry has now revealed that these regulatory processes differ markedly among the different cell types of the reproductive tract. In addition there is new immunocytochemical evidence of the importance of stromal-epithelial interactions in steroid hormone action. Steroid-induced epithelial growth and/or differentiation can occur in tissues where steroid receptors are only detectable in the stromal cells. In this review, immunocytochemical evidence for the nuclear localization of estrogen and progestin receptors, for the variation in receptor regulation in different cell types and for the possible hormone action will be presented. All data will be based on experimental studies of the reproductive system of male and female nonhuman primates.

125I-Luteinizing Hormone (LH) binding to soluble receptors from the primate (Macaca mulatta) corpus luteum: Effects of ethanol exposure

In vitro exposure to alcohols unmasks additional binding sites for gonadotropin in cell/membrane preparations of the corpus luteum of rhesus monkeys. In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30 min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for 125I-human luteinizing hormone (hLH) than were available in particulate preparations (p less than 0.05). However, the soluble receptors displayed a 3-fold lower affinity for 125I-hLH (p less than 0.05). Conditions which enhanced LH binding to particulates, i.e., 1-8% ethanol at 25C, decreased specific 125I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors.

Laboratory instrument interface system (LIIS): a unit for the acquisition, temporary storage and transfer of data to a microcomputer

A simple method of interfacing clinical and research laboratory equipment with a microcomputer is described. A four-channel buffer system has been constructed which stores data generated from laboratory instruments and then transmits the data directly to a microcomputer. The system is highly flexible with respect to the type of laboratory equipment and model of computer that can be interfaced, and it allows for virtually automatic data acquisition and analysis.