Studies on reproductive endocrinology of the female bonnet monkey (Macacu radiata) hormonal regulation of follicular maturation, ......

Abstract

Ovarian function in primates is unique in that, during any given cycle, a single follicle among a cohort of follicles develops into a large mature follicle, which subsequently ovulates and forms a corpus luteum. If fertilization occurs, corpus luteum function is prolonged until the placenta assumes this role. The progression from the initiation of follicular growth, selection of a dominant follicle, formation of a Graafian follicle, ovulation, corpus luteum formation, and eventual luteolysis leading to menstruation is cyclical and regulated by gonadotropins, gonadal peptides, and steroid hormones. Although considerable information is available on primate menstrualcycle regulation, little is known about the specific requirement of FSH in promoting follicular growth at different maturation stages. Since much progress has been made using FSH antibodies in rodents, a similar approach was adopted here to investigate the role of FSH in follicular maturation in the bonnet monkey (Macaca radiata), used as a primate model. Ovulation and subsequent corpusluteum formation are critical because corpus luteum function determines menstrualcycle length. The regulation of corpus luteum function has been a longstanding question in primate reproductive endocrinology. The present thesis examines the relative roles of LH, FSH, estrogen, and prolactin in corpus luteum regulation using specific hormone antagonists. Implantation of the fertilized ovum (blastocyst) in primates occurs around day 7-9 postfertilization. During this periimplantation window, peripheral sexsteroid concentrations increase significantly. While progesterone is well established to be essential for pregnancy maintenance, the role of estrogen during implantation is less clear. The present thesis investigates the need for estrogen for implantation and early postimplantation survival of the blastocyst. The thesis is divided into two sections. Section A deals with follicular maturation and corpus luteum function. Section B focuses on estrogen requirements for pregnancy establishment.

SECTION A Chapter 1: Introduction A comprehensive review of the hormonal regulation of primate ovarian function. Chapter 2: Colony Standardization and Antiserum Preparation This chapter describes the standardization of a colony of female bonnet monkeys:

Monitoring menstrual cycles Measuring steroid hormonal profiles Establishing ovulation timing via laparoscopy Selecting fertile animals based on successful past pregnancies

Normal cycle length: 27.3 ± 1.2 days Hormonal characteristics:

Estrogen surge: Day 9-10 Midcycle LH and FSH surges: Day 10-11 Ovulation: Day 11-12 Progesterone peak: Day 18-20 Decline in progesterone by day 28 menstruation

FSH antiserum was raised in a male bonnet monkey using purified ovine FSH. It was rigorously characterized to ensure specificity (no crossreactivity with LH or unrelated proteins). The antiserum’s binding affinity and capacity were determined using Scatchard analysis.

Chapter 3: Determination of Minimum Effective Antiserum Dose The goal was to determine the smallest dose able to neutralize endogenous FSH for 24 hours. Key findings:

Minimum effective dose: 25 µL antiserum for a ~5 kg monkey FSH neutralization on specific cycle days produced distinct outcomes:

Day 5: Delay of estrogen surge Day 6: Normal estrogen surge, but a second surge on day 14 (new follicle recruitment) Day 7: Most sensitive stage dominant follicle failed, new follicle selected on day 16 Day 8-12: No effect on follicle development or ovulation

Follicles thus become independent of pituitary FSH 48-72 hours before ovulation.

Chapter 4: Induction of a Short Luteal Phase (SLP) Defect Deprivation of FSH on day 6 or 7 shortened the luteal phase, maximally on day 7:

Normal luteal length: 18.2 ± 1.5 days After FSH deprivation: 10.8 ± 1.9 days

Despite normal ovulation, the corpus luteum formed was defective. Fertile females treated with FSH antiserum on day 7 for three cycles did not conceive, but conceived normally once treatment ceased. This model is excellent for testing inhibinbased contraceptives.

Chapter 5: Hormonal Regulation of Corpus Luteum Function Findings:

Chorionic gonadotropin increased both progesterone and estrogen, but estrogen remained elevated longer. Luteectomy abolished both hormones corpus luteum is the primary estrogen source. Decline in progesterone:estrogen ratio cycle termination estrogen may be luteolytic. Tamoxifen extended luteal phase and supported progesterone, suggesting inhibition of estrogen’s luteolytic action. LH deprivation abrupt fall in progesterone cycle termination. FSH deprivation during luteal phase no effect on CL function. Manipulation of prolactin had minimal or transient effects prolactin is not essential for luteal function in primates.

SECTION B Chapter 6: Literature Review on Estrogen in Primate Pregnancy Progesterone is essential for implantation and pregnancy maintenance. The role of estrogen, however, is less certain compared to rodents and rabbits.

Chapter 7: Experiments on Estrogen Requirement for Pregnancy Establishment Using tamoxifen (a potent antiestrogen):

Oral tamoxifen at 3 mg/kg/day (day 18-30 or day 16-20) prevented pregnancy in 90-100% of fertile bonnet monkeys. Progesterone levels remained normal failure was not due to luteal insufficiency. Exogenous progesterone did not rescue pregnancy. A single dose of 25 mg on day 14 was as effective as a 5day lowdose course.

This demonstrates a definitive requirement for estrogen in primate implantation and shows tamoxifen’s potential as a postovulatory contraceptive.

Chapter 8: Summary and Conclusions The thesis provides major insights into:

FSH’s critical role in early follicular maturation Hormonal regulation of corpus luteum function Essential role of estrogen in implantation and early pregnancy Potential contraceptive applications of FSH neutralization and estrogen antagonism