Studies on conadal function in the rabbit effect of specific FSH Deprivation on ovarian function

Abstract

Rabbit is a typical example of an induced ovulator where ovulation is not a cyclic activity. Instead, ovulation occurs only after coital stimulation. This stimulus, via the neural reflex pathway, has been shown to induce release of a surge of luteinizing hormone (LH) within 1–2 h, and ovulation occurs about 10 h thereafter. Alternately, ovulation can also be induced in an adult virgin female rabbit at any given time by administration of an ovulatory dose of LH. This would then mean that the ovary of the adult female rabbit is always endowed with follicles in a readily ovulable state. While considerable information is available on spontaneous ovulators with regard to the role of follicle-stimulating hormone (FSH) in the process of follicular maturation, this area of regulation of follicular development by gonadotropin remains mostly unexplored in the case of induced ovulators. Since significant inroads have been made in understanding the mechanism of FSH regulation of follicular maturation in rodents and the sub-human primate by using specific FSH antibodies, it was felt worthwhile to use a similar approach to attempt understanding the follicular maturation process in the rabbit. The study has been extended to cover the need for FSH, if any, in regulating luteal function of the rabbit. Chapter I, serving as an introduction, provides an overall survey of the literature currently available on the subject.

Chapter II Chapter II describes the methods adopted to standardize the rabbit model system. This involves the establishment of endocrine profiles in adult female rabbits before and after receiving an ovulatory trigger of LH injection (day 1). Within 2 h of LH administration, serum progesterone levels rise sharply in the manner of a surge and decline to basal levels by 4 h. The process of ovulation and formation of a functional corpus luteum is indicated by an increase once again in serum concentration of progesterone, reaching peak values by day 11. Progesterone declines to basal values by day 15, the period day 1–15 contributing to pseudopregnancy in the rabbit. Unlike progesterone, estrogen levels do not show dramatic change after the ovulatory trigger of LH. The pattern of FSH secretion was also studied. The levels start increasing by 12 h of the ovulatory trigger and reach peak values by 24 h, returning to basal values within 48 h. Chapter II also describes procedures used for characterization of the FSH antisera (a/s) employed to neutralize endogenous circulating FSH. An a/s to ovine FSH raised in a monkey was characterized using well-standardized techniques: (a) removal of non-specific antibodies to sheep serum and tissue proteins by treatment with an insolubilized normal sheep serum polymer; (b) removal of the major contaminant LH antibodies present in the FSH a/s by immunoaffinity chromatography using CNBr-activated Sepharose coupled to ovine LH (oLH). The treated a/s was confirmed to lack contaminating antibodies by checking binding with radioiodinated rabbit LH while ensuring its ability to bind rabbit FSH remained intact. Further, the antibody was characterized for: (c) cross-reactivity with rabbit FSH; (d) affinity to bind rabbit FSH and the number of rabbit FSH binding sites via Scatchard analysis; (e) minimum effective dose to neutralize circulating rabbit FSH and duration of efficacy.

Chapter III Experiments were conducted to determine the effect of a single injection of FSH a/s on basal production of progesterone and estrogen as indices of follicular activity. Levels of both steroids were significantly reduced over a 6 h period following injection of 0.2 ml of FSH a/s. The ability of an ovulating dose of oLH to initiate ovulation in follicles deprived of FSH support was next examined. Earlier data showed that 0.2 ml of FSH a/s deprives animals of FSH support for at least 18–20 h. Ovulation and corpus luteum formation were assessed by progesterone levels on day 11 post-LH injection. FSH neutralization up to 18 h blocked ovulation and luteinization. Follicles already at the ovulable stage were highly sensitive to even short FSH deprivation (6 h). Follicles that needed to reach the ovulable stage appeared to progress despite partial lack of FSH but failed once they reached the sensitive stage. The development period to ovulability seems very short (18–24 h). This acute dependence is unlike that in rodents and primates. This suggested that short-term lack of FSH affects granulosa cell competence to respond to LH. Granulosa cells from a/s-treated rabbits showed drastically reduced progesterone production even when stimulated with added gonadotropins.

Chapter IV Granulosa cells isolated from a/s-treated rabbits were examined for changes in gonadotropin receptor concentration-none were observed. However, cells incubated with dibutyryl cAMP showed significantly reduced progesterone production. Forskolin also failed to stimulate progesterone production. This suggested defects in steroidogenic machinery. Additionally, granulosa cell cAMP levels were markedly lowered and could not be elevated to control levels by oLH. Thus, deprivation of FSH affects both cAMP production and the steroidogenic response to cAMP.

Chapter V This chapter extends knowledge of LH and FSH in regulating luteal function. Earlier studies using X-irradiated ovaries suggested that rabbit corpora lutea are initially estrogen-independent and later become estrogen-dependent. In the present study, administration of LH a/s to rabbits with functional corpora lutea caused sharp decline in serum progesterone but only marginal reduction in estrogen. LH a/s plus estradiol restored progesterone levels. FSH a/s administered in early (18–24 h) or mid-luteal (days 6–8) phases showed that FSH deprivation during mid- but not early luteal phase reduced progesterone levels. Absence of FSH receptors in corpus luteum suggested an indirect role, likely via regulation of follicular estrogen production. Indeed, FSH but not LH stimulated aromatase activity in granulosa cells.

Chapter VI Using cathepsin-D as a lysosomal marker, LH deprivation for 96 h increased enzyme activity in corpora lutea. LH a/s plus estrogen normalized activity, indicating estrogen can maintain structural integrity. Estrogen is also known to reverse follicular atresia-related lytic changes. Luteolysis in cycling hamsters was studied by measuring luteal function and lysosomal enzyme activities. LH receptor levels and progesterone peaked at diestrus and declined thereafter. Lysosomal enzyme activities were highest at diestrus, indicating luteolysis. Increased activity reflected true increase in enzyme content.

Chapter VII A general discussion of results in context of existing literature is provided. Chapter VIII This chapter provides the general summary and conclusions of the study.