Reproduction: Gonadotropin-Releasing Hormones

Abstract

GnRH substituents have wide-scale applications in treating diverse categories of reproduction-related cancers that are associated with the release of gonadotropin from the pituitary. We hypothesized that GnRH antagonists also suppress OSE proliferation which is the common cause of OEC’s in humans and primates. Marmoset monkey was used as an animal model owing to similarity with the human reproductive hormonal cycle, and Antarelix was chosen as a peptide antagonist due to its safety and efficacy in clinical application. Administration of Antarelix at zero time and on the 5th day of ovulation in animals, whose ovulation period was synchronized, dramatically reduced OSE proliferation detected a significant reduction in BrdU staining. The extent of proliferation was also determined using the same marker in cycling and pregnant animals in the OSE layer in proximity to stroma, antrum and CL. The staining was performed at early, mid and late follicular and luteal phases and early pregnancy. The epithelia overlaying the large antrum at pre-ovulatory stage and CL at the early luteal phase displayed the maximum staining, while hardly a few cells atop the stroma reacted with the marker. The morphology of the epithelial cells in contact with stroma was typical resting cuboidal shaped with tight attachment to basement layer. The cells above CL were prominently squamous type with loosened attachment, and those over large antral follicles were flattened and disorganized. Apparently, the cell multiplication and intercellular adhesion were distorted in the layers above CL and antrum, which correlates with their proliferative activity. Considering a role of granulosa cells in the differentiation of large antral follicles and subsequent vascularization and steroidogenesis at the onset of CL formation, the proliferative index was measured in these cells. Growing follicles of cycling and early pregnant animals exhibited very high degree of multiplication. From the results, we conclude that luteogenesis contributes to OSE proliferation either by involving factors like VEGF and/or mitogens, β-estradiol, and inflammatory agents, or this is just a consequence of mechanical damage of the OSE layer to accommodate large antrum and CL. Further, GnRH antagonists administered at any time during the follicular phase can alleviate the OSE proliferation.

Introduction

Gonadotropin-releasing hormones (GnRH’s) are decapeptides produced from specialized neurosecretory cells in the hypothalamus, targeting the pituitary-gonadal axis. Over 20 different forms of GnRH in vertebrate and protochordate species have highly conserved structures. In mammals only two forms are known, GnRH I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and GnRH II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2). GnRH I is a typical pituitary controlling hormone, with affinity to the pituitary seven-transmembrane domain G protein (Gq/11)-coupled receptors. After binding, GnRH I modulates membrane phospholipase C (inositol phosphate) and calcium channel, and consequently, it stimulates LH synthesis and secretion. Direct control over FSH secretion has not been unequivocally proven. An elevated plasma gonadotropin level regulates the overall reproductive process, especially the gonadal steroidogenic and gametogenic functions (Millar, 2005). GnRH II, besides pituitary gonadotropin stimulation, also imparts in neural development and sexual arousal in the hypothalamus region. The GnRH I and II receptors are also found in almost all types of normal and cancerous epithelial cells, including those present in gonads (Millar et al. 2001). The extra-pituitary function of these receptors in these tissues is not fully understood, but evidence suggests that the GnRH I and II signal transduction pathways in epithelial tissues are different from pituitary cells (Emons et al. 2003). Interestingly, GnRH II receptors in these epithelia have conserved regions of GnRH I and binding of the two GnRH’s to their receptors can be mutually exclusive (Manputha et al. 2007). Studies suggest that GnRH II has a role in neuromodulation, regulation of energy intake, and suppression of cell proliferation and apoptosis by triggering a G-protein (Gi) mediated signal pathway in the target cells (Szkudlinski 2007).

During the last four decades, several thousand peptides and non-peptide GnRH agonists and antagonists have been synthesized and tested for clinical efficacy. These compounds, which are generally GnRH analogs, competitively bind to pituitary GnRH receptors and by two distinct mechanisms down-regulate gonadotropin synthesis and secretion. Within 7-14 days of a GnRH agonist’s administration, a “surge” of gonadotropins and steroidal hormones, known as “flare”, is followed by a rapid fall in their serum concentrations. On the other hand, the antagonists lower the hormone concentrations from the beginning. Both groups of synthetic analogs also down-regulate the expression of GnRH receptors and initiate the internalization of the cell surface receptors to the nuclear membrane (Herbst, 2003). This further hinders the binding of GnRH to pituitary cells. Clinically, the agonists/antagonists are used as male contraceptives and for the treatments of prostatic, breast and endometriotic cancers, uterine fibroids, precocious puberty, premenstrual and polycystic syndromes, and infertility (Emons et al. 2003; Millar 2005; Hayden 2008). During the ’90s Antarelix (Ac-D-Nal-D-Cpa-D-Pal-Ser-Tyr-D-Hci-Leu-Lys-(iPr)-Pro-D-Ala-NH2) has been formulated and extensively used as a polypeptide GnRH I antagonist (Deghengh et al. 1993). The advantage of this drug has been its high water solubility, modest allergy and histamine stimulation and high potency.

Because the similar pattern of serum β-estradiol, progesterone, testosterone, LH/CG during estrous/anestrous and pregnancy phases to that of the human being, nonhuman primates are used as experimental models to rectify hormone-related reproductive problems like anovulatory infertility (Steinetz, Randolph & Mahoney, 1995; Abbott et al. 2004). Among primates too, the reproductive physiology of Old World (e.g. rhesus; Macaca mulatta) and New World (marmosets; Callithrix jacchus) monkeys differs. Some ubiquitous features of marmoset distinct from higher primates are: 1) multi-ovular species with ovulatory cycles of ~28 days, comprising short follicular (8-9 days) and long luteal (19 days) phases, 2) ovulation rates ranging from one to four follicles per cycle (Gilchrist et al. 2001), 3) pituitary gonadotropes secret preferentially chorionic gonadotropin (CG) and not LH, 4) LH receptors bind to CG and course of action of two hormones is similar, and 5) ovarian senescence is more subtle and do not resemble menopause in women. Like humans, the follicular phase is characterized by a low level of progesterone, rising estradiol and typical pre-ovulatory “surge” of LH, albeit LH “surge” is not tightly regulated by GnRH. Marmoset ovaries contain a multitude of small antral follicles and towards end of the follicular phase; two or three antral follicles enlarge and go on to ovulate. There exists clear morphological and size distinction between large ovulating and small non-ovulating antra (Gilchrist et al. 2001). Ovarian epithelial proliferation is believed to be primarily regulated at the level of gonadotropins, which stimulate follicular estradiol production. As a mitogen, β-estradiol and other localized factors viz. insulin-like and hepatocyte-growth factors (IGF, HGF) trigger proliferative response in the post-ovulatory OSE layer at the sites of rupture. Poly-ovulating mammals like marmosets have repeated ovulation and therefore repeated rupture and healing of OSE layer. These features accelerate OSE cell multiplication and transformation to neoplastia at the rupture sites. Notwithstanding, gonadotropin-associated incidences of ovarian cancers are rarely reported in marmosets, even though EOC affects only primates and no other mammals. It is likely that about 10-fold higher progesterone concentration in pregnant marmosets compared to other primates (Steinetz, Randolph & Mahoney, 1995) help suppress the OSE tumorigenesis in these animals.

Antarelix GnRH antagonist has been used in marmosets to suppress LH/CG secretion from pituitary gland and the follicular and luteal development was examined in such treated animals (Taylor, Hillier & Fraser, 2004). It was found that upon such treatment the serum β-estradiol rapidly falls down and as a result proliferation of granulosa cells in antrum and CL diminishes. More importantly follicular vasculature remained underdeveloped and the resultant follicle’s growth and differentiation get stunted and ovulation becomes unsuccessful. The GnRH antagonists may also suppress the mammalian OSE proliferation and tumorigenesis in dualistic manner. First, it may do so by inhibiting LH/CG secretion so that follicular development and ovulation gets affected. Hence the number of re-epithelization events is reduced and OSE stays non-proliferative. Second, the antagonists may bind directly to OSE cells’ GnRH receptors and impart anti-proliferative response through some unknown downstream signaling pathway. Another action could be that reduced LH-controlled steroidogenesis brings down the production of localized pro-proliferative mitogens (β-estrogen) in granulosa cells and other inflammatory agents which cause genetic damage to the epithelial cells.

According to the gonadotropin theory of ovarian cancer, a high level of gonadotropins at menopause has a role in the development of epithelial ovarian cancer. In this paper, we test the hypothesis that a standard GnRH antagonist’s administration would suppress endogenous gonadotropin, resulting in inhibition of OSE proliferation in marmoset. We also hypothesize that changes in the ovarian environment during the ovarian cycle (follicular and luteal phase) and at early pregnancy might have an influence on the activity and morphology of the OSE cells. To our knowledge, this is the first report on effect of a GnRH antagonist on marmoset OSE proliferation.

Discussion

In this investigation, the anti-proliferative response of a GnRH antagonist was examined on OSE cells in estrous marmoset monkeys. Before this experiment the CL was artificially degenerated and luteal phase was prematurely stopped by adding a prostaglandin F substitute towards the end of estrous cycle, which is a common practice to synchronize the follicular phase in cycling marmosets (Steinetz, Randolph & Mahoney, 1995). Luteolysis was verified as sharp reduction in serum β-estradiol concentration (Taylor, Hillier & Fraser, 2004). Towards the end of luteolysis the steroidal hormones reach their minimum concentrations and under LH/CG influence the steroidogenesis begins in pre-antral granulosa and antral granulosa and theca cells. Blocking LH/CG release at zero time by Antarelix (0-10 days) in this study would have arrested the development of primary follicles and consequent ovulation must have been averted. Further, β-estradiol synthesis also might have gone down as Antarelix would inhibit steroidogenesis (Taylor, Hillier & Fraser, 2004). At 5th day blockage (5-10 days) some gonadotropin concentration would have built up within the first 5 days which is sufficient to grow follicles up to the pre-antral stage. Thereafter, LH “surge” which takes place after 5 days in the follicular cycle necessary for ovulation and CL formation must have been hindered. Since in marmosets, large antra are differentiated only after 6 days (Gilchrist et al. 2001), possibly in this treatment regime, neither large antrum would be differentiated nor CL would be formed. In this situation, only the small antral follicles are expected to develop up to the pre-ovulatory stage and then start to regress into atresia (Taylor, Hillier & Fraser, 2004). The depressive effect of Antarelix on OSE proliferative activity was clearly discernable regardless of the time of administration. It is proposed that pre-ovulatory large antrum and its derived CL may be responsible for OSE proliferation in the contact regions. This conclusion is derived from observed OSE proliferation was suppressed when the drug was administered on 5th day of the follicular cycle when only pre-ovulatory follicles were not able to be differentiated. There could be several explanations of Antarelix’s OSE anti-proliferative effect which may function in isolation or in combination: 1) reversal of LH/CG up-regulation of localized growth factors like IGF and HGF which are responsible for mitogenic action through their anti-apoptotic effect on OSE, 2) inhibition of gonadotropin-induced steroidogenesis in antral follicles and CL would minimize production of mitogenic β-estradiol in granulosa cells, and consequently OSE proliferation would be averted, 3) GnRH antagonists result in skew in frequency of antral follicles (Taylor, Hillier & Fraser, 2004), and apparently this would reduce ovulation frequencies and resultant epithelial rupture, and thereby prevent the neoplastic growth of OSE, and 4) this compound may bind to GnRH I/ GnRH II receptors in OSE cells itself and inhibit all downstream pathways which involve mitogen activating protein (MAP), p38 and the extracellular signal regulate kinase (ERK-1/2) responsible for mitogenic proliferation (Millar et al. 2001; Mamputha et al. 2007).

From the present results, it appears that during the estrous cycle BrdU staining of the OSE layer increases towards the end of the follicular phase, when it is in contact with large antral follicles and decreases from the mid luteal phase when CL starts to recede. The highest staining was seen in CL at early luteal phase of estrous and during early pregnancy. Low staining of OSE, layering stroma suggests that the resting OSE cells at early follicle development or those which are not in the vicinity of the CL or antrum are not profusely dividing. Since OSE proliferation was associated with late follicles and CL at all reproductive phases it can be predicted that events leading to luteogenesis contribute towards OSE proliferation whereas the reverse process, luteolysis, suppresses the proliferative response. During luteogenesis remarkable changes take place in the granulosa and theca cells of growing follicles in the late follicular phase, notably vascularization, angiogenesis and steroidogenesis. Interestingly, these changes are regulated by LH/CG (Taylor, Hillier & Fraser, 2004; Duncan et al. 2009). Angiogenesis is tightly regulated by an endogenous cellular factor called vascular endothelial growth factor (VEGF). In granulosa cells of pre-antral follicle, VEGF expression is up-regulated most likely due to LH, and a high cellular level of this factor is maintained up to the luteal phase controlled by LH/CG. In CL the same granulosa cells transform to granulosa-lutein cells responsible for steroidogenesis. It was therefore a matter of interest to examine whether or not the angiogenic and steroidogenic granulosa cells proliferate at late follicular and early luteal phases. The granulosa cells of growing follicles were heavily stained suggesting that these cells are multiplying and transforming to micro-vascular cells. We propose that these rapidly multiplying granulosa cells secrete some mitogenic substances, notably β-estradiol, to influence the surrounding OSE layer to proliferate.

OSE cell morphology is believed to be another marker of proliferative activity (Auersperg et al. 2001). It has been suggested that squamous and cuboidal forms of cells represent respectively the groups that have not or have undergone post-ovulatory proliferation. OSE cells tend to assume columnar shapes when they form clefts and inclusion cysts. These cells are the primary targets for mitogenic and inflammatory action leading to tumorigenic growth. Besides, the secretion of adhesive proteins like cadherins determines the association of the OSE layer with the underneath basement membrane. We found that non-proliferative cuboidal and tightly attached OSE cells were present away from the ovulation sites. The proliferative squamous cells with very loose attachment were discernable over CL and disarranged flattened cells were present in the OSE layer just before ovulation. Some of these cells might transform to columnar cells if inclusion cysts or clefts are formed after ovulation.

Since OSE cells and underneath pre-antral follicles’ granulosa cells during 2nd week of pregnancy also displayed positive BrdU staining, it is obvious that the proliferation is not necessarily related to only post-ovulatory rupture of the OSE layer. It was rather surprising to detect OSE proliferation in proximity to CL and granulosa cells of pre-antral follicles during early pregnancy. This was because the progesterone concentration must have been high enough to suppress any proliferative action. Earlier we have shown an anti-proliferative role of progesterone whether produced during pregnancy or is given as contraceptive pills. It is reasonable to assume that despite progesterone’s presence, CL influences the OSE proliferation and this attribute could be due to some factor that supersedes the hormone action. The data also suggests that as pregnancy advances the proliferation becomes diminished because the regression of CL eliminates the factor which overrides the progesterone effect.

So far the results indicate that marmoset luteogenesis rather than folliculogenesis seems to influence OSE proliferation and luteolysis and pregnancy reverse this process. In primates, LH/CG is necessary for CL development, which induces numerous paracrine regulations for luteal development including VEGF that controls angiogenesis and vascularization. (Duncan et al. 2009). As LH concentration starts to recede, the natural luteolysis advances starting from mid-luteal phase. During luteolysis the LH level falls and all its stimulatory actions like angiogenesis and steroidogenesis invert. Besides, luteal cells also produce interleukins, prostaglandins and factors like capsase-3 to induce apoptosis and other mechanisms to degenerate CL (Fraser et al. 2006). If CL is rescued due to pregnancy, CG produced from conceptus takes over the LH functions. In this investigation, the granulosa proliferation in CL during early luteogenesis and at early pregnancy appears to be the result of LH/GC-mediated up-regulation of VEGF and other luteinizing factors and steroid production, a process that is reversed during luteolysis. It remains to be seen whether in primates local ovarian environment influence OSE proliferation or it is simply the mechanical ablation of OSE layer to accommodate large antrum or CL that triggers the renewed multiplication and neoplastic transformation. In this context, it is noteworthy that in rhesus monkeys brushing the OSE layer at any reproductive phase is a good enough reason to induce proliferative repair (Wright et al. 2007).

In conclusion, BrdU immunoreactivity was detected at the ovulation site before and after ovulation and a lower number of BrdU–stained cells were found to be distributed randomly around the ovary, suggesting that the OSE proliferative activity cyclically takes place depending on the ovarian events that occur underneath the OSE during the ovarian cycle. GnRH antagonist treatment resulted in complete inhibition of OSE cells proliferation. Further study is needed for investigating the mechanisms in which the GnRH antagonist exerts its effect on OSE cells, whether by direct or indirect pathway.

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