Mary B. Zelinski-Wooten

Developmental potential of embryos produced by in-vitro fertilization from gonadotrophin-releasing hormone antagonist-treated macaques stimulated with recombinant human follicle stimulating hormone alone or in combination with luteinizing hormone

We previously demonstrated, in luteinizing hormone (LH)-deficient macaques, that follicular growth and maturation occurred with administration of exogenous (recombinant human) follicle stimulating hormone (r-hFSH) alone, and that the oocytes recovered fertilized at a notably higher rate than their counterparts from animals receiving both r-hFSH and r-hLH (Zelinski-Wooten et al., 1995). Here, the developmental potential of embryos produced from animals treated with r-hFSH alone or in combination with r-hLH was evaluated. Embryos (n = 127) were cryopreserved, thawed and either co-cultured on buffalo rat liver cells until the hatched blastocyst stage or transferred to synchronized recipients. Although embryos from each treatment group demonstrated a similar ability to develop to hatched blastocysts with a definitive inner cell mass, a significant difference was seen in cryosurvival (56 versus 78%) and in developmental rate to the hatched blastocyst (12 versus 10 days) between embryos from the r-hFSH alone and the combination group respectively. Pregnancies resulted following oviductal embryo transfers in both groups, with corpus luteum rescue occurring on days 12-16 of the luteal phase. In summary, r-hFSH alone during the pre-ovulatory interval is adequate for the gametogenic events required to produce embryos that develop either in vitro or in vivo; however, exposure to r-hLH may improve embryo viability and the rate of development.

Steroid reduction during ovarian stimulation impairs oocyte fertilization, but not folliculogenesis, in rhesus monkeys * †

OBJECTIVE To test the hypothesis that steroids locally modulate and may be required for normal follicular function and gametogenesis in primates, the effects of steroid reduction during gonadotropin-stimulated folliculogenesis was studied in rhesus monkeys. DESIGN Animals received human FSH (hFSH; days 1 to 6) and hFSH+human LH (hLH; day 7) to promote multiple follicular growth, and then received hCG (day 8) for ovulatory maturation. Four animals received trilostane (3 beta-hydroxysteroid dehydrogenase inhibitor) on days 1 to 8 or no inhibitor (controls; n = 4). Follicles were aspirated 34 hours after hCG. MAIN OUTCOME MEASURES Follicular growth, serum E2, P, and pregnenolone, oocyte nuclear maturity, and IVF. RESULTS Trilostane markedly reduced E2 to levels as low as 7% of controls throughout the follicular phase. Pregnenolone was 66-fold greater during trilostane treatment relative to controls. In both groups, P was at baseline during follicular stimulation but was reduced for 72 hours after hCG in trilostane-treated animals. Despite E2 suppression, follicular growth and oocyte nuclear maturity were unaltered by trilostane. Trilostane hindered the fertilizability of metaphase II oocytes (15%) in three of four animals compared with controls (65%). Metaphase I oocytes that required > 8 hours to complete meiosis in vitro failed to fertilize in the same three of four receiving trilostane relative to controls (31%). CONCLUSIONS Follicular growth and oocyte meiosis did not require high or increasing E2 levels. Levels of follicular products other than E2 may be of value in determining the progress of ovarian stimulation protocols. However, the acquisition of oocyte competence for fertilization may require steroids.

Progesterone Promotes Oocyte Maturation, but Not Ovulation, in Nonhuman Primate Follicles Without a Gonadotropin Surge

Abstract During the periovulatory interval, intrafollicular progesterone (P) prevents follicular atresia and promotes ovulation. Whether P influences oocyte quality or maturation and follicle rupture independent of the midcycle gonadotropin surge was examined. Rhesus monkeys underwent controlled ovarian stimulation with recombinant human gonadotropins followed by a) experiment 1: an ovulatory bolus of hCG alone or with a steroid synthesis inhibitor (trilostane, TRL), or TRL + the progestin R5020; or b) no hCG, but rather sesame oil (vehicle), R5020, or dihydrotestosterone (DHT). In experiment 1, the majority of oocytes remained immature (65% ± 20%) by 12 h post-hCG. However, the percentage of degenerating oocytes increased (P < 0.05) with TRL (42% ± 22% vs. 0% controls), but was reduced (P < 0.05) by progestin replacement (15% ± 7%). By 36 h post-hCG, the majority of oocytes in all three groups reached metaphase II (MI). In experiment 2, no evidence of follicle rupture was observed in the vehicle, R5020, or DHT groups. Despite the absence of hCG, a significant (P < 0.05) percentage of oocytes resumed meiosis to metaphase I in R5020- (41 ± 9) and DHT- (36 ± 15) but not vehicle- (4 ± 4) treated animals. Only oocytes from R5020-treated animals continued meiosis in vivo to MII. More (P < 0.05) oocytes fertilized in vitro with R5020 (40%) than with vehicle (20%) or DHT (22%). Thus, P is unable to elicit ovulation in the absence of an ovulatory gonadotropin surge; however, P and/or androgens may prevent oocyte atresia and promote oocyte nuclear maturation in primate follicles.

Maturity and fertility of rhesus monkey oocytes collected at different intervals after an ovulatory stimulus (human chorionic gonadotropin) in in vitro fertilization cycles

In rhesus monkeys undergoing ovarian stimulation for in vitro fertilization (IVF), a midcycle injection of human chorionic gonadotropin (hCG) substitutes for the LH surge and induces preovulatory oocyte maturation. The time interval between injection and oocyte collection, ideally, allows for the completion of oocyte maturation without ovulation, which would reduce the number of oocytes available for harvest. To evaluate the influence of this time interval on oocyte parameters following hCG administration, we conducted a series of gonadotropin treatment protocols in 51 animals in which the interval from hCG administration to follicular aspiration was systematically varied from 27 to 36 hr. Follicle number and size, evaluated prior to hCG administration by sonography, did not vary significantly or consistently with preovulatory maturation time. Oocytes were harvested by laparotomy or laparoscopy, and scored for maturity before insemination. The percentage of mature, metaphase II (MII) oocytes at recovery increased significantly with increasing preovulatory time and was inversely proportional to that of metaphase I (MI) oocytes. However, oocyte yield tended toward a progressive decrease with increasing preovulatory maturation times from a high of 27 oocytes at 27 hr to a low of 17 oocytes/animal at the 36 hr time interval. Fertilization levels declined significantly from a high of 50% at 27 hr to a low of 30% at 36 hr. Thus, although higher percentages of mature oocytes were recovered at the longer time intervals, optimal oocyte/embryo harvests were realized after the shorter time intervals (27 and 32 hr) and are most compatible with the goal of achieving high yields of fertile oocytes and embryos following gonadotropin stimulation in rhesus monkeys.

Androgen receptor mRNA expression in the rhesus monkey ovary.

Immunocytochemical detection of androgen receptors (ARs) in several compartments of the macaque ovary, including the germinal epithelium, follicle, and corpus luteum, suggests a role for androgens in modulating ovarian function via the classical receptor-mediated pathway. To examine AR mRNA expression in the rhesus monkey ovary, total RNA was isolated from whole ovaries, the germinal epithelium-enriched cortical and medullary compartments of the ovary, and corpora lutea from early (d 3–5), mid (d 6–8), midlate (d 10–12), and late (d 13–15) stages of the luteal phase of the menstrual cycle. RNA was also obtained from luteinized granulosa cells from monkeys receiving gonadotropin treatment to stimulate the development of multiple ovarian follicles. After reverse transcription of total RNA using oligo-dT as a primer, polymerase chain reaction (PCR) was used to amplify a unique 329 bp segment of the monkey AR hormone-binding region. Reverse transcriptase (RT)-PCR products of the expected size were detected in all ovarian and control tissues. Sequence analysis of the AR cDNA from the macaque ovary revealed 99% nucleotide homology and 100% predicted amino acid homology to the cDNA for the hormone-binding region of human AR. Northern analysis demonstrated the presence of a major AR mRNA species at 9.5 kb in corpus luteum, luteinized granulosa cells, and prostate, with additional bands detected in the corpus luteum and prostate at 7.9 and 3.4 kb, respectively. A sensitive RNase protection assay was used to examine AR mRNA levels in ovarian tissues and showed AR mRNA expression throughout the life-span of the corpus luteum. Thus, detection of AR mRNA in the primate ovary, including the periovulatory follicle and corpus luteum, supports the concept that these tissues are targets for receptor-mediated androgen action during the menstrual cycle.

Assisted fertilization and nuclear transfer in mammals

PART I. ASSISTED FERTILIZATION Developments in Animal Reproductive Biotechnology Advances in Animal in vitro Fertilization Control of Oocyte Nuclear and Cytoplasmic Maturation In vitro Oocyte Maturation: an Aspects Measurement of Reproductive Potential in the Human Female Imaging Technology in Assisted Reproduction Intracytoplasmic Sperm Injection or Conventional Fertilization to Maximize the Number of viable Embryos The Production of Viable Human Biastocyts: Evolution of Sequential Culture Systems Embryo Transfer: oncise and Practical Summary of the Techniques Cryopreservation of Mammalian Embryos, Gametes and Ovarian Tissue: rent Issues and Progress Satellite and Transport in vitro Fertilization The Assisted Reproductive Laboratory: lity Assurance and Continuous Quality Improvement in a Regulatory Environment PART II. NUCLEAR TRANSFER Somatic Cell Nuclear Transplantation in Cattle Nuclear Modifications and Reprogramming After Nuclear Transfer Application of Nuclear Transfer in Exotic or Endangered Species Assisted Fertilization and Nuclear Transfer in Nonhuman Primates Cloning and Nuclear Transfer in Humans

Individualized gonadotropin regimens for follicular stimulation in macaques during in vitro fertilization (IVF) cycles.

Follicular stimulation was compared in macaques receiving sequential gonadotropin treatment which was terminated after seven, eight, or nine days depending on the time required to attain preselected criteria of follicular maturation. Although estradiol levels and follicle sizes varied, the number of follicles and oocytes/animal, oocyte nuclear maturity, IVF rates and progesterone levels during the luteal phase were similar among groups. Reducing the duration of gonadotropin treatment to individualize follicular stimulation regimens does not compromise follicle or gamete quality.

Steroid receptors and action in the primate follicle

Studies localizing steroid receptor (R) proteins and their mRNAs to the primate ovary are consistent with a local role for progesterone (P) and androgen (A) in modulating follicle growth and/or maturation via classic R-mediated pathways, but data in support of estrogen (E) action remain equivocal. Investigations of the hypothesis that steroids play a pivotal role in folliculogenesis or gametogenesis in macaques receiving inhibitors of steroid enzyme synthesis and in women with congenital steroid enzyme deficiencies reveal that (a) antral follicle growth, maturation, and luteinization, as well as oocyte meiosis, do not require high or increasing E levels; (b) elevated A to E ratios are detrimental to the gametogenic functions of the primate follicle, but do not alter growth and maturation of antral follicles; and (c) ovulation and luteinization of the mature follicle are dependent on local P actions. The genomic actions of steroids likely vary between compartments and with the developmental state of the follicle; however, potential nongenomic actions of steroids in follicles remain largely undefined. Future advances will rely on identification of discrete biochemical, morphological, and functional correlates of steroid hormone action in the somatic and gametogenic compartments of the primate follicle throughout its life cycle.

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.

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).