Hamish M. Fraser

Differential Expression of Estrogen Receptor-α and -β and Androgen Receptor in the Ovaries of Marmosets and Humans

Abstract Estrogens and androgens are essential for the maturation of the ovarian follicle and normal fertility in the female. We have used antibodies specific for both forms of estrogen receptor (alpha [ERα] and beta [ERβ]) and androgen receptor (AR) to investigate the pattern of receptor expression in ovaries obtained from women and from a New World primate, the Common marmoset (Callthrix jacchus). On Western blots, three antibodies directed against different peptides within human ERβ all recognized recombinant human (h) ERβ but did not bind to recombinant hERα. The ERβ protein was extracted from human ovary and prostate and marmoset ovary. In marmoset and human ovaries, ERβ protein was detected in the nuclei of granulosa cells in all sizes of follicle (both before and after formation of the antrum), and it was also detected in thecal cells, corpora lutea, surface epithelium, and stroma. In contrast, ERα protein was not detected in the nuclei of granulosa cells in preantral follicles, was low/absent from stromal and thecal cells, but was expressed in granulosa cells of antral follicles and in the surface epithelium. The pattern of expression of AR protein more closely resembled that of ERβ than ERα. In conclusion, three independent antibodies have demonstrated convincingly that ERβ is expressed in a wide range of cells in the primate ovary. Granulosa cells in preantral follicles could contain ERβ:β dimers. In antral follicles, however, ERα is also expressed, and the formation of homo- or heterodimers containing ERα may influence the pattern of gene activation within these cells.

Angiogenesis During Follicular Development in the Primate and its Inhibition by Treatment with Truncated Flt-1-Fc (Vascular Endothelial Growth Factor TrapA40)1

The aims of this study were to 1) quantify changes in angiogenesis during follicular growth in a primate model; 2) investigate the molecular regulation using in situ hybridization of vascular endothelial growth factor (VEGF), its receptor, Flt-1, the angiopoietins (Ang-1 and Ang-2), and their receptor, Tie-2; 3) elucidate the role of VEGF in follicular angiogenesis by blocking its action by treatment with a soluble truncated form of the Flt-1 receptor, (VEGF Trap(A40)). Changes in angiogenesis were quantified using bromodeoxyuridine to obtain a proliferation index, and CD31 immunocytochemistry to visualize endothelial cell area. Percentage of proliferating endothelial cells was calculated by double labeling for bromodeoxyuridine and CD31. Vascularization was first observed in follicles containing four granulosa cell layers. A significant increase in proliferation in the thecal layer was observed from the early to late secondary stage, and dual staining showed that 25% of proliferating cells were of endothelial cell origin. VEGF messenger RNA (mRNA) was expressed in granulosa cells with an increase of grain density from late secondary to tertiary follicles. Ang-1 was weakly expressed in the theca of tertiary follicles. Ang-2 mRNA was not detected in any follicles. The mRNA for the Flt-1 and Tie-2 receptors was localized in endothelial cells of the theca. Unexpectedly, Tie-2 mRNA was also found in granulosa cells of early follicular stages and its translation was confirmed by immunocytochemistry. VEGF trap treatment for 3 days resulted in an 87% decrease of proliferation in the theca of secondary and tertiary follicles, a reduction in endothelial cell area and a marked decline in Flt-1 mRNA expression. Granulosa cell proliferation also decreased. These results show that onset and establishment of the follicle vasculature takes place early during follicular development. The ability of VEGF trap treatment to severely restrict follicular angiogenesis establishes that VEGF is the major regulator of this process in the primate ovary.

Hypothalamic pulse generators

Publisher Summary This chapter describes pulsatile hormone secretion and analyzes the mechanisms underlying the neural organization of this phenomenon in context of oxytocin and luteinizing hormone releasing hormone (LHRH) secretion. It also discusses five concepts— (1) oxytocin and LHRH are both released from the hypothalamus in pulses superimposed upon a continuous or intermittent low level of secretion; these two modes of secretion can produce separate actions, or one might govern the response to the other; (2) the synchronous generation of action potentials at a very fast rate within a population of peptidergic neurones provides the neural substrate for the release of a hormone pulse; (3) the control of interpulse interval is determined by events within the brain, though the interval generator may not reside within the neurons that secrete oxytocin or LHRH; (4) amplitude modulation of pulsatile secretion can relate to different levels of electrical activation within the brain and/or to differences in the responsiveness of the target tissues that transduce the pulsatile signal; and (5) opioid peptides inhibit the secretion of both oxytocin and LHRH. This involves an inhibition of stimulus-secretion coupling within the nerve terminals, and possibly an inhibition of synaptically mediated events that impinge upon the cell bodies of the peptidergic neurons within the hypothalamus.

Angiogenesis and its control in the female reproductive system

The rapid, controlled and cyclical nature of angiogenesis in the female reproductive tract suggests that interference with this process should provide a novel approach to manipulation of reproductive function. Many factors involved in the regulation of angiogenesis have been identified, and the possibility of stimulating or inhibiting these paracrine control mechanisms is being addressed using current advances in the development of angiogenic and anti-angiogenic compounds. Studies with animal models indicate that the normal processes of folliculogenesis, ovulation and corpus luteum function in the ovary, and the control of menstruation and implantation in the endometrium could be profoundly influenced by manipulation of angiogenesis. Novel therapeutic agents targeted to the angiogenic pathway may also have a wide range of applications in pathological processes in the reproductive tract such as cancer, endometriosis, fibroid growth, and ovarian hyperstimulation syndrome.

Angiogenesis in the corpus luteum

The corpus luteum (CL) is a site of intense angiogenesis. Within a short period, this is followed either by controlled regression of the microvascular tree in the non-fertile cycle, or maintenance and stabilisation of the new vasculature a conceptual cycle. The molecular regulation of these diverse aspects is examined. The CL provides a unique model system in which to study the cellular and molecular regulation of angiogenesis. Vascular endothelial growth factor (VEGF) has been found to have a major role in the CL. By targeting its action at specific stages of the luteal phase in vivo by antagonists, profound inhibitory effects on luteal angiogenesis and function are observed.

Hemochorial Placentation in the Primate: Expression of Vascular Endothelial Growth Factor, Angiopoietins, and Their Receptors Throughout Pregnancy

Abstract Vascular development and its transformation are necessary for successful hemochorial placentation, and vascular endothelial growth factor (VEGF), angiopoietins, and their receptors may be involved in the molecular regulation of this process. To determine the potential role of these putative regulators in a widely studied primate, the common marmoset, we investigated their mRNA expression and protein location in the placenta throughout pregnancy using in situ hybridization, Northern blot analysis, and immunocytochemistry. VEGF was localized in decidual and cytotrophoblast cells, and its highest expression was found in the maternal decidua. The Flt receptor was exclusively detected in the syncytial trophoblast with increasing expression in placentae from 10 wk to term. Soluble Flt (sFlt) was also detectable by Northern blot analysis. KDR receptor expression was restricted to mesenchymal cells during early placentation and to the fetoplacental vasculature during later placentation. KDR expression increased throughout pregnancy. Angiopoietin-1 (Ang-1) was localized in the syncytial trophoblast, being highly expressed in the second half of gestation. Ang-2 mRNA localized exclusively to maternal endothelial cells, and was highly expressed in 10-wk placentae. The Tie-2 receptor was found in cytotrophoblast cells and in fetal and maternal vessels. High Tie-2 levels were detected in the wall of chorion vessels at 14-wk, 17-wk, and term placentae. These results suggest that the processes of trophoblast invasion, maternal vascular transformation, and fetoplacental vascular differentiation and development are regulated by the specific actions of angiogenic ligand-receptor pairs. Specifically, 1) VEGF/Flt and Ang-1/Tie-2 may promote trophoblast growth, 2) VEGF/KDR and Ang-1/Tie-2 may support fetoplacental vascular development and stabilization, 3) sFlt may balance VEGF actions, and 4) Ang-2/Tie-2 may remodel the maternal vasculature.

Effect of Neonatal Gonadotropin-Releasing Hormone Antagonist Administration on Sertoli Cell Number and Testicular Development in the Marmoset: Comparison with the Rat

Abstract The primary purpose of this study was to establish whether Sertoli cells proliferate in the neonatal period in the marmoset monkey (Callithrix jacchus) and whether administration of a long-acting GnRH antagonist (GnRHa) during this phase induced any transient or permanent effects on Sertoli cell number or on any other aspect of testicular development. Male marmoset co-twins (n = 9) were treated during Weeks 1–14 with either vehicle or GnRHa. Four sets of co-twins were examined at Weeks 18–22 (start of infancy) and 5 sets in adulthood (92+ wk), and Sertoli cell number was determined using either the nucleator or optical disector methods; other testicular morphometric analyses (e.g., germ cell volume, Leydig cell volume) used standard point-counting. Data for the marmoset were compared with that obtained in similarly treated rats. Sertoli cell number in marmosets treated neonatally with GnRHa was reduced by 35% compared with that of controls at Weeks 18–22 but was comparable to control values in adulthood. However, seminiferous epithelium volume was reduced significantly in adult marmosets treated neonatally with GnRHa, and there was a tendency for reduced germ cell volume per Sertoli cell. In the same animals, there was significant expansion of the interstitium and an increase in Leydig cell volume per testis when compared with co-twin controls; a similar increase in Leydig cell volume was evident in adult rats treated neonatally with GnRHa. Comparison of Sertoli cell numbers in 6 infantile (18–24 wk) and 10 adult marmosets showed that adult numbers of Sertoli cells were present by the start of infancy but, unlike rats, marmosets were still able to replicate Sertoli cells beyond this period. However, marmoset Sertoli cells supported only ∼20% of the germ cell volume supported by rat Sertoli cells, indicative of poor efficiency of spermatogenesis, as shown previously in the human. This finding, together with the demonstration of a temporal pattern of Sertoli cell replication similar to that in the human, supports the use of marmosets as a model for human male testicular development and function.

THE SECRETION, MEASUREMENT, AND FUNCTION OF A TESTICULAR LHRH‐LIKE FACTOR

In this paper we have demonstrated the presence and secretion within the testis of a factor that is immunologically distinct from LHRH, but that has receptor-binding and bioactive features similar to LHRH. The major source of this LHRH-like factor is probably the Sertoli cell and, as its site of action appears to be restricted to the Leydig cells, it is possible that it is one of the means by which the Sertoli cell has been postulated to regulate the Leydig cell. All of the available evidence suggests that the action of the LHRH-like factor on the Leydig cell is inhibitory, and it may mediate some or all of the negative effects on the Leydig cell of LH and hCG. The precise role of the LHRH-like factor, particularly under physiological conditions (i.e., low level stimulation with LH), remains to be determined, but the availability of methods for its extraction, measurement, and inactivation should enable this problem to be resolved within the near future. Moreover, the recognition that Leydig cell function may be regulated by local factors within the testis should open up new avenues of research that may lead to improved methods for the regulation of fertility in the male.

Angiogenesis in the primate ovary

The ovary is distinctive in undergoing cyclic changes in angiogenesis that play a critical role in the normal functioning of the female reproductive system. The current paper describes the use of the marmoset monkey as an in vivo model in which the cellular and molecular regulation of angiogenesis in the ovary can be investigated and the effects of manipulation of angiogenic factors elucidated. The studies are based on quantifying changes in blood vessel area and endothelial cell proliferation, monitoring changes in expression patterns of putative angiogenic regulatory factors and targeting these factors by antagonists in vivo. Quantification of endothelial cell proliferation shows that angiogenesis commences in the pre-antral follicle, increases with follicular development and becomes intense in the early corpus luteum. Vascular endothelial growth factor (VEGF), a principal angiogenic factor, is synthesized by the developing follicle and corpus luteum. Administration of specific antagonists in vivo for selected periods of the ovulatory cycle shows that inhibition of VEGF results in a marked decrease in endothelial cell proliferation in the follicle and is accompanied by a decline in granulosa cell proliferation. Inhibition during the early or mid-luteal phase results in a marked suppression in luteal angiogenesis, failure of development of the microvascular tree and suppression of luteal function. Manipulation of angiogenesis should be a novel approach to either promoting or inhibiting the normal processes of folliculogenesis, ovulation and corpus luteum function.

Vascular morphogenesis in the primate ovary

Ovarian function is dependent on intense cyclical vascular morphogenesis and regression. The molecular and cellular pathways involved in the generation of new capillary networks in the ovary are now being elucidated. Focussing on the marmoset, the course of angiogenesis at different stages of follicular maturation and in the corpus luteum throughout the cycle and in early pregnancy have been quantified and major progress has been made in the evaluation of the role of vascular endothelial growth factor (VEGF). To study the physiological role of VEGF in follicular and luteal angiogenesis in detail, VEGF was inhibited during defined stages of the cycle in vivo. VEGF antagonist administered throughout the follicular phase of the cycle resulted in a marked decrease in endothelial cell proliferation in developing antral follicles, accompanied by a decline in granulosa cell proliferation, restriction of follicular growth and inhibition of ovulation. An outstanding feature in the ovary is the intense angiogenesis that occurs during the early luteal phase. VEGF inhibitors markedly suppressed this angiogenesis, resulting in a marked restriction in the development of the microvascular tree and suppression of plasma progesterone. These studies showed that VEGF is essential for normal follicular and luteal angiogenesis and function, and demonstrated how luteal angiogenesis in particular could serve as a sensitive bioassay for putative angiogenic antagonists. Antagonists of VEGF are potent tools for investigating the role of angiogenic factors within the ovary and may have applications to the treatment of reproductive disorders characterised by alterations in normal vascular structure or function.