The tale of follitropin receptor diversity: A recipe for fine tuning gonadal responses?

Molecular and Cellular Endocrinology 260–262 (2007) 163–171

The tale of follitropin receptor diversity: A recipe for fine tuning gonadal responses?夽 M. Ram Sairam ∗ , P. Suresh Babu 1 Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec H2W 1R7, Canada Received 20 September 2005; accepted 21 November 2005

Abstract The original concept (dogma) of a single FSH receptor entity coupling to Gs protein to activate adenylate cyclase and producing cAMP as second messenger appears inadequate to explain pleiotropic actions of the hormone. The identification and expression of alternatively spliced gonadotropin receptors, suggest that alternative splicing could serve as a mechanism for creating receptor diversity. Studies focused on sheep and mouse gonadal tissues show that the single large gene of ∼250 kb is a modular structure whose pre-mRNA undergoes alternative splicing creating several subtypes (at least four FSH-R1 to R4 identified to date). With segments of the N-terminus that are identical different topographies are generated by differing carboxyl termini. The same gene thus produces receptor types with different motifs that can display dominant positive, dominant negative, growth factor/cytokine type and potentially soluble binding protein features. Functional relevance is shown by modulation of receptor variants during hormonal stimulation. Presence of equivalent segments of the gene in the human and bovine suggests conservation and predicts similarity in structures and function. Thus, the complex cellular biology of follitropin receptors that may interact differently with polymorphic forms (glycosylation variants) of FSH represents an intricate scheme to regulate hormone signaling. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Ovary; Testis; Alternative splicing; Signaling; Gene regulation

“Nature does nothing without a purpose” “If at first the idea is not absurd . . . then there is no hope for it”—Albert Einstein 1. Introduction: so few genes and myriad of proteins The number of genes in the human genome and other mammals is steadily decreasing from previous projections of more than 100–150,000 in the pre-genome era to as few as 23,000 estimated or identified by the end of 2004 when refined sequence data became available (International Human Genome Sequencing Consortium, 2004). How could such a small number that is about the same as the tiny flowering plant Arabidopsis or slightly more than the lowly worm Caenorhabditis elegans, account for the complexity as some 500,000 to a millions plus versions of potential proteins are believed to be functional in 夽 ∗ 1

The terms follitropin and FSH are used interchangeably in this article. Corresponding author. Tel.: +1 514 987 5582; fax: +1 514 987 5585. E-mail address: [email protected] (M.R. Sairam). Current address: Lundbeck Research USA, Inc., Paramus, NJ 07652, USA.

0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.11.052

mammalian systems? The discovery of split genes and RNA splicing (Sharp, 2005) has provided the framework necessary for understanding the mystery underlying the paradoxes. Thus, it is now estimated that approximately 60% of human genes undergo alternative splicing to create dozens or hundreds of proteins from a single gene and together with a variety of post translational modifications, vastly increases the diversity of the proteome that can be encoded by a limited set of genes. The understanding of mechanisms and the regulatory proteins (splicing factors) involved in controlling alternative splicing of the pre mRNA and post translational modifications, remains a big challenge and will provide the basis for defining dynamic physiological events and their aberrations in pathology. While the process of alternative splicing of the same gene producing different hormonal proteins in unrelated tissues with examples of calcitonin and the calcitonin gene related peptide was well known in endocrinology, few had given thought to the possibility of multiplicity of gonadotropin receptors until the first reports of cloning of the pig LH receptor cDNA (Loosfelt et al., 1989; Vuhai-Luuthi et al., 1992) and complexity of amplicons generated during the amplification of the LH receptor in the hormone primed rodent ovary (Aatsinki et al., 1992). Our

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investigations first directed at the cloning of ovine FSH receptor (FSH-R) from testicular cDNA library revealed a variety of related transcripts coding for structurally related but yet different receptor forms with distinct topographical features. This article highlights some of these findings in the context of ovarian and testicular developments and discusses its potential validity to human and other mammals. We postulate that such receptor diversity in concert with hormone glycosylation variants that will arise during the reproductive cycle or aging (Ulloa-Aguirre et al., 1995, 2003) could provide additional mechanisms for regulation of gonadal functions. 1.1. Pleiotropic actions of gonadotropins Gonadotropins and FSH in particular have pleiotropic actions on target cells in the different stages of the ovary and testis stimulating cellular growth, differentiation and steroidogenesis. These coordinated actions culminate in the development/maturation of oocyte in a single large follicle that is destined for ovulation in primates and other species or the daily production of millions of sperm during spermatogenesis in the testis. How does the ovarian/testicular responses to gonadotropins change with development, cell cycle or stage of spermatogenesis? For example, what makes gonadotropins induce mitotic activity during early stages of follicular growth (Hirshfield, 1986) which then switches to activation of other events in cellular differentiation (Richards, 1994; Richards et al., 1998)? It is also intriguing that despite the presence of similar number of FSH-Rs per cell, granulosa cells from small antral follicles produce least cyclic AMP in response to FSH than do the cells at more advanced stages of growth (Richards, 1994). Similarly, FSH-R numbers and responses also vary with the stage/cycle of spermatogenesis (Kanagasniemi et al., 1990). Despite considerable evidence that cyclic AMP functioned as a major second messenger of LH or FSH action in ovarian and testicular cells, not all responses could be explained, implicating other second messengers and signaling pathways. These early reports arose well before the use of cloned cDNAs of receptors came in to vogue for addressing mechanisms (Cooke, 1990; Sharma et al., 1994; Quirk and Reichert, 1988; Grasso and Reichert, 1990). Thus, it is not unreasonable to question if all these actions are mediated by a single receptor. If not, what other mechanisms are utilized to account for their pleiotropic effects? With the same blood circulation accessible to all target cells in the ovary and testis, such events or stage specific actions of the hormone cannot be regulated by changing FSH levels but this could well be controlled by hormone receptor numbers/types and other intracellular events. By combining receptor diversity and functional forms with subtle changes in the nature of the hormone (e.g., variable glycosylation) (Ulloa-Aguirre et al., 2003) delivered to the cell and the transducing machinery, one could visualize a fine tuning mechanism for cell signaling. 1.2. Molecular basis for a variety of follitropin receptors The existence of multiple mRNAs for glycoprotein hormone receptors first revealed during the cloning of pig LH-R cDNA

(Loosfelt et al., 1989) has been extended to other LH-R, FSHR (Simoni et al., 1997) as well as the TSH-R from different species (Mishrahi et al., 1994). These mRNAs ranging from 1.2 to 7 kb for gonadotropin receptors revealed by Northern analysis or RT-PCR evaluation is present in both sexes. However, such analyses may not reveal the complete picture of all transcripts as they are directly dependent upon the probes used for Northern’s or primers selected for amplification. We utilized a complementary approach of analyzing whole cDNA libraries constructed from adult gonadal tissues for exploring different types of receptors. Such studies also offer another useful alternative to the practice of mutagenesis of a single receptor cDNA to establish functional relevancy of discrete residues or domains (see Moyle et al., 2005). Structural differences in the FSH-R gene as studied mostly for the classical Gs coupled receptor may not drastically alter signaling functions except for certain activating or inactivating mutations (Aittom¨aki et al., 1995; Simoni et al., 1997; Haywood et al., 2002; Huhtaniemi and Themmen, 2005). As compound heterozygous mutations in certain cases produce early ovarian failure (Beau et al., 1998; Touraine et al., 1999) additional studies in hormone resistant patients in different parts of the world would be more informative. Some recent studies have uncovered evidence that certain discrete polymorphisms do confer differences in the threshold required to elicit hormonal responses in women (Perez Mayorga et al., 2000; Sudo et al., 2003). Notwithstanding these observations, alternative splicing is a logical explanation for the existence of different receptor transcripts that are formed by usage of cassette-exon mode and 3 -alternative acceptor sites. In some instances incomplete processing (splicing) could lead to retention of certain intronic sequences causing premature termination. A composite of some of these structures (although not exhaustive) characterized during our cloning studies (Fig. 1A) illustrate these features. In the expectation that structures similar to those we have identified (in the ovine) might exist in other species, a revised nomenclature is proposed to designate previously cloned FSH-R variants. As indicated, the R1 encodes a putative mature protein of 675 amino acids, with the other three variants (R-2–4) having 653, 242 and 117 residues, respectively. Interestingly, a clone of FSH-R variant designated 5–10 (hFSH R4 in Fig. 1A) from human testis (Kelton et al., 1992) also contains exons 1–4 similar to the sheep R4 type but its C-terminus is different. The value of examining transcripts in a whole library is evident from the discovery of novel R2 and R3 types which would not have been detected by an RT-PCR approach. Their origin is of interest as it adds to potential functional relevance of unidentified gene segments. The 3 -end of these two cDNAs is identical from the point of divergence from the cDNA coding for the classical R1 receptor. Apparently similar mechanisms that include same polyadenylation signals and length of the poly A tail are used during processing of these two transcripts. Due to splicing at two different locations, the same DNA sequence produced different carboxyl termini for the R2 and R3 receptor types (Fig. 1B). Hence we have concluded that this segment of the FSH-R gene must participate as an additional exon in the final assembly of receptors with different motifs (Sairam et al., 1997).

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Fig. 1. (A) Generation of four receptor motifs from a single oFSH R gene. This FSH-R gene is estimated to be ∼250 kb. Arrow head in each indicates the point at which their C-termini differ. The mature R protein is shown for each receptor type. R1 = dominant positive type; R2 = dominant negative type; R3 = growth factor type I; R4 = soluble type. There may be other forms which remain to be fully characterized. (B) Same DNA segment of the FSH-R gene producing two different carboxyl termini in two receptor motifs. oFSH-R3 transcript arises by splicing the 392 bp segment shown at the eighth exon creating a single transmembrane domain. When the same segment is spliced at nucleotide #1925 in exon 10 (same as R1) the dominant negative receptor R2 is produced. The extreme carboxyl terminal is different from both R3 and R1.

1.3. Different structural motifs in follitropin receptors? Structural differences in known forms of alternately spliced FSH-R transcripts are of two types. Some variants are missing exons in the extracellular domains while others have changes at the 3 end introducing a different characteristic for the receptor. The functional consequences of receptor forms lacking certain exons but identical to the wild type in other respects are not completely known. In the monkey LH-R, the lack of 27 amino acids contributed by exon 10 that is present in all other species

does not affect function (Zhang et al., 1997). In contrast to this hFSH-receptor lacking exon 9 (62 residues), is apparently inactive (Dankbar et al., 1995). The structural differences at the C-terminal of FSH-R types reveal new features that could be of physiological significance. The persistence of type R4 mRNA as a major transcript throughout development in sheep testis (Yarney et al., 1997a) and being quantitatively higher than R1 is suggestive of some new function. As the four exons present in R4 account for about 40% of the extracellular domain of full-length type R1, a

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recombinant protein expressed in E. coli binds FSH via its ␣subunit (Khan et al., 1997). Thus, it is possible that gonadotropin subunits expressed with in the testis (Markkula et al., 1995), might be involved in interacting with entities of the R4 type or similar structures as part of a paracrine mechanism. Such non-membrane proteins could also partially account for buffersoluble receptors previously detected in testis extracts (Dias and Reichert, 1982). The possibility that R4 type binding proteins could also lend some significance to previous but contentious FSH binding found in germ cells (Orth and Christensen, 1977). The extreme C-terminus of oFSH-R4 contains a motif RYLFKR↓WRNRIH that is susceptible to potential cleavage by a proprotein convertase enzyme called PC5 that is localized in Sertoli/granulosa cells and acts on type IV protein precursors (Lusson et al., 1993). As PC5 generates anti-mullerian hormone from its precursor (Nachtigal and Ingraham, 1996) in the Sertoli cell, we suggest that a similar action on FSH-R4 is a possibility. If this event occurs, a regulated liberation of a charged hexapeptide such as WRNRIH from the Sertoli cell/germ cell could form part of a novel dialogue in the intercellular communication. The FSH-R3, with a single transmembrane segment and lacking the heptahelical region of R1 implicated in coupling to heteromeric G proteins, is a characteristic that is typical of receptors for cytokine/growth factors. The growth promoting effects mediated by this receptor are independent of cAMP/PKA-pathway but dependent upon Ca2+ and PKC pathway (Babu et al., 2000; Touyz et al., 2000). A report on presence of an R3 type of receptor in transformed mouse ovarian surface epithelial cells and its apparent linkage to Ca2+ signaling raises potential implications of growth factor functions of the hormone in ovarian tumorigenesis (Li et al., 2005). The FSH R3 but not R1 or R2 also contains the sequence PVILSP that is a potential consensus motif (PXnS/TP, where X is a basic or neutral residue) for phosphorylation (unpublished data) by MAPK (Gonzalez et al., 1991). Designation of R3 as type I receptor is in accord with the proposal of growth factor structure motif of glycoprotein hormones (Lapthorn et al., 1994) including hFSH (Fox et al., 2001). Recent X-ray crystallographic data of the hormone along with the extracellular domain of the receptor suggests that domains such as in FSH-R3 could generate productive interactions (Fan and Hendrickson, 2005). Thus, the R3 receptor entity has enough structural information including majority of the leucine repeats of the extracellular domain necessary for FSH recognition (Braun et al., 1991) and expression on the cell surface (Sairam et al., 1997; Babu et al., 2000). The ability of FSH-R3 to enhance H3 -thymidine incorporation into DNA upon transfection (Sairam et al., 1997) and selective activation of MAPK pathway via PKC and Ca2+ dependent pathway (Babu et al., 2000) is consistent with the proliferative actions of FSH in gonadal cells. Activation of components ERK pathway and other actions in ovarian cells from immature rats (Salvador et al., 2001; Cottom et al., 2003) is also in accord with well-established growth effects of FSH but such studies cannot clearly discriminate the potential role(s) of individual FSH-R types. In any case, the actions of FSH on immature ovarian follicular cells causing DNA synthesis at cellular stages when there is no action of the hormone on cAMP synthesis/or

steroidogenesis (Richards, 1994; Roy and Greenwald, 1986; Delidow et al., 1990; Miro and Hillier, 1996) might implicate the R3 type receptor as the primary participant in signaling. Evidence that hormone priming (Babu et al., 2001) in rodents of a type similar to that used in ovarian stimulation in women favors the production of an R3 type of receptor over FSH-R1 (Fig. 2A and B) lends credibility to the growth factor function of the hormone via the R3 type receptor. While this does not diminish or preclude the role of FSH-R1 in such events, the preferential use of one over the other is a distinct possibility. Thus, we have postulated in our working model that splicing events control the predominance of one type of receptor over the other during ovarian development and cycle (Fig. 2). Further validation and understanding the regulation of these events in the cycling ovary represents a new challenge in reproductive biology. The FSH-R2 (Fig. 1A) is the only known member of the glycoprotein hormone receptor family alternatively spliced in the coding segment that produces the intracellular cytoplasmic domain. This change reduces the available number of potential phosphorylation sites from 12 to 4, an alteration that may affect both structure and function (Quintana et al., 1994; Krishnamurthy et al., 2003). Although there is no data on the relative abundance of R2 in the testis, dynamic changes in FSH-R along the length of the seminiferous tubule could be part of the mechanisms for differences in cAMP generation. 1.4. Follitropin variants in other species In addition to the data derived from cloning of variants in sheep and mouse gonads, comparison of the published sequence of the human and bovine genomes also reveal the existence of putative structures similar to FSH-R2 and R3. Alignment of these predicted structures reveal similarity at both the genome level in organization of the putative 11th exon and peptide sequences (Fig. 3). Among the three species there is 64% identity in the carboxyl terminus. As the successful isolation of ovarian FSH-R3 in sheep (Babu et al., 1999) and mouse (Babu et al., 2001), was facilitated by hormone priming, collaborative studies with Dr. Catherine VandeVoort of UC Davis in CA using granulosa cells from FSH primed female monkeys are in progress to assess the existence and potential regulation of the monkey FSH-R variant transcripts. 1.5. Signaling pathways and crosstalk Examples of modulation of signaling by means of variant receptors coupling to specific G proteins and producing different second messengers are known (Hulme, 1990; Namba et al., 1993). Despite numerous mutagenesis studies on the structurefunction of gonadotropin receptor genes, only few investigations have assessed the potential function of alternatively spliced transcripts. We believe that these are crucial to our understanding the physiological significance of the phenomenon of splicing as they directly address the functional status of these novel structures and potential interactions among different receptor entities.

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Fig. 2. Hormonal regulation of FSH-R splicing in the developing mouse ovary. Expression of FSH-R1 and R3 using receptor specific primers for RT-PCR of ovaries from 21-day-old immature female mice treated with a single injection of equine chorionic gonadotropin. Hormonal (FSH) regulation of FSH-R pre-mRNA splicing in the immature Mouse ovary. eCG primed ovaries were evaluated for specific mRNAs and protein (R3 only). (A) Multiplex RT-PCR using R specific primers. Note that R3 is higher than R1. (B) Western blot of R3 protein in the above ovaries 1-saline, 2–24 and 3–48 h after hormone priming. Right panel: predicted balances in the regulation of FSH-R3 and R1 during ovarian follicle development and function. We postulate that receptor related dynamic events occur during normal ovarian growth. When early and rapid growth is required R3 action is likely to dominate; and when a robust steroidogenic function becomes necessary in a more differentiated state R1 action is expected to prevail.

Such interactions could result in potentiation of hormone action in vitro as reported for the LH-receptor (Vuhai-Luuthi et al., 1992). On the other hand, attenuation of hormone action by sequestration of the ligand from circulation by soluble receptor proteins remains a distinct possibility (Khan et al., 1997; Osuga et al., 1997). The participation of growth hormone binding protein generated from alternate splicing of the receptor gene in pathophysiology of growth represents a clear example of the importance of such mechanisms (Baumann, 1995) which remain to be fully explored for the gonadotropin receptors. Functional assessments of the FSH-R types provide some new clues to their significance. Designating the R1 type as the full-length receptor appears to be justified as this form activates adenylate cyclase following FSH binding. This presumably is coupled to signaling mechanisms via Gs proteins. While some

have argued that GPCRs can couple to multiple mechanisms (Gudermann et al., 1992; Laugwitz et al., 1996) others contend that members of the leucine rich G-protein coupled receptor family could each interact with only specific subclasses of many structurally similar proteins occurring in a given cell (Watson and Abbott, 1990). However, even when such a variety of coupling can be demonstrated in vitro for the full-length receptor, interpretation should be carefully guarded owing to excessive levels of over expression of receptors on the cell surface, as gonadal cells may have only 5000–6000 receptors per cell. Moreover, even these small numbers could vary with the spermatogenic/ovarian cycle. At high receptor densities as is common in transfection studies promiscuity in receptor-G protein interactions compromise specificity (Gudermann et al., 1992; Laugwitz et al., 1996). Therefore, such coupling differences

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Fig. 3. Exon–intron arrangement of the human FSH-R gene and comparisons of the carboxyl termini of the FSH-R3 in different species. (Top) The hFSH-R is located on chromosome 2 and exon–intron arrangements are shown from data derived from cDNA cloning and genomic cloning reports. The predicted exon 11 is based on sequence comparison of human genome data with the nucleotide sequence of ovine FSH-R2 and R3. Bottom panel compares putative peptide sequences of human FSH-R3 and bovine FSH-R3 with oFSH-R3. For all three sequences there is 64% identity (84% for ovine and bovine FSH-R3). The carboxyl terminus of mouse FSH-R3 is identical to that of oFSH-R3 (Babu et al., 2001 and unpublished data).

could be specified by regions of the receptor that are slightly different and located on the inner side of the plasma membrane. Thus, in direct contrast to R1 type, failure of the FSH R2 to stimulate cyclic AMP accumulation may be a result of differences in C-terminal structure (Yarney et al., 1997b; Sairam et al., 1996). Potential coupling to G␣i found in co-expression studies suggest dominant negative function (Sairam et al., 1996). We can interpret previous reports of Pertussis toxin augmentation of FSH action in cultured Sertoli cells as resulting from the inhibition of R2 type receptor action on G␣i (Grasso and Reichert, 1990; Huhtaniemi et al., 1989; Monaco et al., 1988). The alterations of G␣i protein mRNA turnover by FSH as reported in Sertoli cells (Loganzo and Fletcher, 1992) could be mediated by a R2 type receptor. Similar arguments for the LH-R are also credible because in ovarian membranes that may contain mixed LH receptor types, LH/hCG activation leads to association of the receptor with both Gs ␣ and Gi ␣ protein (Rajagopalan-Gupta et al., 1997). The only notable difference between R1 and R2 types of FSH receptors is the shortening of the carboxyl terminus in the latter. Although it has been shown that the third intracellular loop contains the primary regions responsible for specificity in G-protein interactions, the C-terminus may act in concert to establish the full efficiency in desired receptor-G protein coupling. Considering that a variety of receptors that inhibit adenylate cyclase have a shorter C-terminus (Conklin et al., 1993), alternate splicing induced modifications of transcripts is a useful regulatory mechanism. As two reports examining rodent FSH-Rs alternatively spliced by deletions of several exons singly or in combina-

tions found neither hormone binding nor any modulating effect on the activity of the R1 type of receptor in co-transfection studies (Tena-Sempere et al., 1999; Kraaij et al., 1998), further investigations on other species are required to resolve differences. 1.6. Novel modalities for modulation Apart from the contributions of alternative splicing of a single gene many investigations have now shown that GPCR signaling exhibit greater diversity and flexibility than previously appreciated. Signal diversity arises from numerous factors, among which are the ability of receptors to adopt multiple ‘active’ states with different effector-coupling profiles, receptor dimerization, ability to interact with non-receptor modifiers (e.g. RAMPs) coupling to non-G-protein effectors (Bomberger et al., 2005). Two recent developments in particular deserve mention, as such studies might help reconcile previous observations on gonadotropin receptors in tissues with more recent studies on single receptor entities. Considering that diversification of the repertoire of GPCRs through oligomerization apparently allows for more complex-ligand relationships (Waldhoer et al., 2005; Park and Palczewski, 2005) studies in gonadal cells that are subject to precise and dynamic cyclic changes will be rewarding. An additional phase of research in this area requires the understanding of intracellular interacting partners that might be recruited following hormone action. Now that some of these proteins are presently being identified in model cells (Dias et al., 2005), questions of receptor homo- and hetero-dimerizations and potential for differential post receptor recruitments in target cells

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at different stages of development and differentiation assume importance. A novel mechanism for FSH receptor modulation reportedly involves sequestration by an LH receptor variant lacking the exon 9 identified in some women (Yamashita et al., 2005). 1.7. Significance of spliced gonadotropin receptor mRNA for gonadal physiology The emerging theme on alternatively spliced gonadotropin receptors as emphasized from new investigations on the FSH receptor extends this concept further to suggest that various motifs can be generated from a single large gene to create receptor diversity. Thus, three genes encoding the gonadotropins (subunits ␣ and ␤ of LH and FSH) and one gene each encoding many receptor variants allow a more diverse repertoire for integrating signal transduction. Multiple receptors that could be subject to variation during cell cycle of ovarian follicular development (Richards et al., 1998) or spermatogenic stage introduce a new experimental paradigm for investigations. The novelty and predictions arising from these concepts make evolutionary sense particularly for FSH and is supported by the current growth factor model of the hormone (Fox et al., 2001). The FSH-R might be expressed with a growth factor type I receptor motif at stages when rapid cell proliferation is required (Fig. 2). A switch in topography and signaling might then occur to fulfill other functions like steroidogenesis that are also controlled by intricate intragonadal autocrine/intracrine mechanisms. Generation of multiple receptor types shows the versatility of the cell to perceive and interpret the hormonal signal in different ways and offers an explanation for the pleiotropic effects of the gonadotropins. The mechanisms involved in maturation of the gonadotropin signal transduction from the receptor to the various transducers are not fully understood and the relative abundance of the alternative transcripts/proteins would be an important dynamic element of the system to determine the final outcome of hormone action. Tools such as specific nucleic acid probes and antibodies will be critical to distinguish and discriminate these various types as only some have been defined. The potential for regulation at multiple sites to achieve signal integration becomes evident when we also add the well-known fact of gonadotropin heterogeneity, including their propensity to display variations in glycosylation (Ulloa-Aguirre et al., 1995) that is critical for signaling (Sairam and Bhargavi, 1985) and modulation of biological activity (Arey et al., 1997). A better understanding of these polymorphic gonadotropins and receptor variant systems might eventually help resolve critical issues related to dominant follicle selection or atresia and pave the way for improved treatment of infertility and understanding of pathology. Acknowledgements Investigations emanating from this laboratory have received support from the CIHR and NCI of Canada. Our apologies for not citing all papers in this area and these can be found in review articles.

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