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Animal Models and their Importance in Undrestanding Human Infertility

The use of genetically engineered animal models of diseases has been revolutionary to the field of experimental pathology, leading to advances in translational research far beyond those in the era of traditional animal models. Particularly, studies on genetically modified mice have been fostering a remarkable leap forward in our understanding of the pathogenesis, physiology and molecular basis of various human disorders, notably those within the field of human infertility.

During reproduction, the complex cascades of cellular interactions have proven the in vitro modelling of this process extremely difficult if not impossible. In such circumstances, mouse models provide an attractive alternative; where they enable us to investigate primary defects in genes of interest, discover new candidate genes, reconfirm the phenotypes of human mutations and track temporal phenotypic changes.


Mice Models for the Study of Gonadotrophin Actions


The hypothalamic-pituitary-gonadal axis (HPG) plays a critical part in regulating male and female fertility. The hypothalamus is the source for Gonadotrophin-releasing hormone (GnRH), which is essential for the stimulation of the pituitary to release gonadotrophins FSH and LH. In females, follicle-stimulating hormone (FSH) stimulates the growth of ovarian follicles and estrogen production; estrogen is essential in uterine preparation for potential pregnancy. When estrogen levels peak, the ensuing “LH surge” instigates ovulation and subsequent progesterone secretion by the corpus luteum. Lack of oocyte fertilisation results in cessation of progesterone production, involution of the corpus luteum and shedding of the uterine lining. In males, leydig cells are under the influence of LH, which allows testosterone biosynthesis, a hormone necessary for normal sperm production.

The complexity of the hypothalamic-pituitary-gonadal axis necessitates the use of in vivo models to define and analyse the functions of this axis. The use of transgenic and knockout mouse models, in which key components of the reproductive axis are selectively eliminated, have been beneficial to our understanding of the transcriptional regulation and hormonal interactions involved in mammalian reproduction. Understanding such aspects of gonadotrophin biology is clinically significant in relation to gonadal physiology and pathophysiology with important implications in fertility management.

Both LH and FSH and their corresponding receptors have been genetically targeted and inactivated in mice, and a comparison of their phenotypes have been made with patients who carry mutations in these genes (Achermann 2002; Allan et al. 2001; Zhang et al. 2001).


Mice Models of Aberrant HPG Function in Males


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Figure 1 | Hormonal regulation of spermatogenesis. The testes are influenced by both endocrine and paracrine factors. Whilst the endocrine regulation of the testis is based on the actions of gonadotrophins LH and FSH, the paracrine regulation of testicular function is deemed reliant on steroids such as testosterone and estradiol; Leydig cells as well as proteins such as inhibin and activin (synthesised by sertoli cells) are responsible for production of these steroids (Cooke & P. T. K. Saunders 2002b).

While newborn Lhr knockout mice (LuRKO) have no marked abnormal gonadal development, their adult male counterparts have underdeveloped seminiferous tubules, heavily reduced germ cell development and blockage of spermatogenesis at the round spermatic stage. The poor differentiation of leydig cells in LuRKO mice relates back to their inability to respond to LH, thus LH signals fail to stimulate testosterone synthesis by leydig cells. Hence due to lack of androgens essential for adult sertoli cell stimulation, the limited germ cell development seen in these mice, must have been a result of indirect FSH action on sertoli cells (Zhang et al. 2001).

Lack of FSH receptor in young male mice (FORKO mice) (Krishnamurthy et al. 2000), leads to underdeveloped testis with marked reduction in number of sertoli cells, this is inline with previous observation that FSH is involved in stimulation of sertoli cell replication before puberty in both mice and men (Cooke & P. Saunders 2002a). Delayed puberty, late round spermatid formation and lowered adult male testosterone levels in the serum are characteristics of FORKO mice. These models, although able to reproduce, are sub-fertile, mainly due to abnormal sperms and defectively compacted DNA (Krishnamurthy et al. 2000). Surprisingly the reduction in testosterone levels observed in FORKO mice occurs in presence of normal circuiting LH concentrations; this hints at possible hindrances in communications between Sertoli and Leydig cells.

To establish the absolute requirement of FSH for testicular development and spermatogenesis, Allan et al. (2001) developed a novel transgenic mouse model. By expressing FSH from a transgene into the hpg mouse, which is gonadotrophin deficient, it was observed that hpg mice with higher FSH levels have elevated serum testosterone and bigger testes, which also contain round and some elongated spermatids (Allan et al. 2001).

These results hint at the ability of FSH to stimulate sertoli cells in the absence of testosterone, which can aid meiosis to complete and spermiogenesis to initiate, but not complete; a phenotype similar to the LuRKO mice described earlier. Although, the primary causes of the phenotypes seen in these mice must be interpreted carefully by taking into account the potential secondary effects that may arise from disruptions in paracrine regulation of testicular function, shown in figure 1.

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Figure 2 | Schematic representation of the in vivo dimerization of the luteinizing hormone receptor (Vassart 2010).

G-protein-coupled receptors (GPCR) are the primary mediators for a variety of stimuli such as hormones. Recent investigations into GPCR function using the mouse luteinizing hormone receptor (LHR) as a model GPCR, has shown that mice with binding/signalling deficient forms of LHR can be rescued to resume normal LH signalling through “intermolecular functional complementation of the mutant receptors” (ller et al. 2010), in other words, defective GPCRs can cooperate with each other to overcome their defects by using the functional elements of each other as a mean for compensation of their mutations. These transgenic studies using LuRKO mice have extended our understanding of the mechanisms that underlie defective LHR signalling, Meanwhile such studies harbour the potential to be translated into the human settings, particularly for restoring male fertility in cases where spontaneous LHR mutations are the cause of infertility.


Mice Models of Aberrant HPG Function in Females


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Figure 3 | Schematic representation of the female HPG axis (Nussey & Whitehead 2001)

Loss of FSH signalling in women leads to infertility (Layman & McDonough 2000). This condition can be modelled in mice by disrupting the Fshr or FSHβ loci. Women with aberrant FSH signalling have normal pre-antral follicle development, but fail to produce antral stage follicles with the ability to ovulate (Saner-Amigh & Halvorson 2010). These women are clinically characterised with sexual infantilism and amenorrhea, characteristics that are phenocopied by their mouse equivalents; FSH knockout mice present with infertility, uterine hypoplasia and pre-antrum stage blockade in folliculogenesis (Kumar et al. 1997; Abel et al. 2000).

A noteworthy finding in the FSH knockout mice was that despite the presence of estradiol in their serum, the mRNAs encoding for enzymes P450 side chain cleavage (an initiating enzyme for steroidogenesis) and aromatase P450 (responsible for the conversion of androgen precursor steroids to estrogens) were down regulated in the ovaries of Fshb KO mice (Burns 2001). This highlights the importance of FSH signalling in the expression of genes important for normal folliculogenesis and fertility.

The elimination of FSH signalling in female knockout mice, is associated with elevated levels of LH (Abel et al. 2000); this surge in circulating LH is more profound in older mice and is thought to be a trigger for the development of ovarian sex-cord stromal tumours, an incidence which occurs in 92% of FSH knockout mice by 12 months of age (Burns 2002). Further investigations into these tumours showed that there is an up regulation of Anti-Müllerian hormone and Sertoli cell markers, as well as loss of regulation in proliferation of granulosa cells (Danilovich et al. 2001).

The fact that men and mice with FSHR null mutations can reproduce, although less efficiently, suggests that while FSH is necessary for optimal spermatogenesis, its role in male fertility is less profound that it is in females, where its is an absolute requirement for fertility.


Mice Models and Chromosomal Defects


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Figure 4 | A representation of the different stages in spermatogenesis. During this process, the mammalian germ cell undergoes mitosis, meiosis and structural remodelling into the mature spermatozoa (cytoplasm is indicated in green, DNA in pink) (Adapted from Cooke & P. T. K. Saunders 2002b).

Genetic defects, namely microdeletions of the long arm of the Y chromosome, are often seen in infertile men with oligozoospermia or non-obstructive azoospermia (Tanaka & Baba 2005). In humans, the Deleted in Azoospermia (DAZ) gene is located on the AZFc region of the long arm of the Y chromosome; this gene is exclusively expressed in germ cells and encodes RNA-binding proteins. Deletion of this gene is considered detrimental to fertility in men.

Despite our comprehensive understanding of the genomic structure of DAZ, its biological functions, particularly those related to the infertility phenotype in men with the AZFc deletion, remains to be elucidated. This is partly due to the absence of DAZ orthologues in mammals other than humans. However, the presence of DAZ paralogues, such as Dazl in mice has been useful in providing clues to the biological functions of DAZ, including its potential role in germ cell development and identification of its downstream RNA targets.

Mice with Dazl null (Dazl-/-) mutations are deficient of any spermatozoa or oocyte and are thus infertile (Ruggiu et al. 1997). An investigation into the testes of 9 day old Dazl-/- mutant mice revealed a strikingly low number of germ cells and showed that type A spermatogonia are the most advanced cell type present in these mutants, a stage where normally preleptotene spermatocytes are present (Schrans-Stassen et al. 2001). Although, the type A spermatogonia seen in Dazl-/- mice are not quiescent and actively proliferate, but Aaligned spermatogia fail to differentiate into A1 spermatogonia, hinting at the importance of DAZL for the differentiation of type A spermatogonia (Yen 2004).

On the basis of this data, we can conclude that DAZ and DAZL are both essential for the development of primordial germ cells and germ cell differentiation and maturation. Thus Dazl can be titled a candidate for an autosomal infertility locus in humans.


Mice Models of Defective Folliculogenesis


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Figure 5 | Stages of folliculogensis and the impacts of Foxl2 inactivation on this process. Folliculogenesis starts with the assembly of primordial follicles in the fetal ovary through association of pregranulosa cells with the emerging oocyte. Germline inactivation of Foxl2 results in complete inhibition of primordial follicle recruitment. Conditional knockout studies in mice have shown that Foxl2 inhibition results in transmogrification of the ovarian follicles into structure that closely resemble testicular tubule. In humans, FOXL2 is associated with reduced follicle reserve due to either disrupted follicle assembly or increase primordial follicle recruitment (Murphy 2010).

Premature ovarian failure (POF) and polycystic ovarian syndrome (PCOS) are both leading causes of female infertility(Boivin et al. 2007). Nonetheless, the causes of these disorders are still unknown in many infertile individuals. However recent studies in mice have broadened our understanding of processes that may go wrong during PCOS and POF. In a recent study by Uhlenhaut et al. (2009) it was discovered that a transcription factor expressed in the ovary, forkhead box L2 (FOXL2), is key for maintaining cellular and structural integrity of ovarian follicles. In mice which the FOXL2 gene is transiently knocked out, the ovaries primarily produce androgens instead of estrogens, highly resembling the phenotype seen in PCOS (Uhlenhaut et al. 2009).

Although the majority of investigations on FOXL2 have been carried out in mice, there is an adequate amount of human data available to signify the effects of defects in FOXL2 mediated processes, which may underlie many types of infertility (Beysen et al. 2009).

FOXL2 is involved in regulating various processes during various stages of folliculogenesis (Figure 1). The ablation of FOXL2 in mice, at specific points of folliculogensis, leads to conditions that resemble the ovarian phenotype seen in infertile women. For example, loss of function mutations of the human FOXL2 gene is associated with the depletion of follicle reserves which leads to premature ovarian failure (Moumne et al. 2008).

Based on the data available from mice knockouts studies and human ovaries with PCOS, FOXL2 appears to have a key role in fate specification of both granulosa and thecal cells. Conditional deletion of FOXL2 for a period of 5 days in female mice leads to disruptions in maintenance of postnatal female ovarian phenotype, such that granulosa cells undergo reverse differentiation to form Sertoli cells and thecal cells increase androgen production (Uhlenhaut et al. 2009). These findings shed light on the importance of FOXL2 in maintaining adult ovarian phenotype.

On the basis of above observations, one can establish a logical link between FOXL2 action and PCOS, since the increased androgen production, reversed differentiation of granulosa cells and the depleted follicular reserves in FOXL2 KO mice highly resembles the phenotype seen in patients with PCOS. On the other hand, premature ovarian failure and androgen-induced hirsutism, are both distinctive consequences of human FOXL2 gene mutations (Méduri et al. 2010). Another striking discovery which has been replicated in human ovary, is that the transcripts of highly expressed FOXL2 targets, such as the orphan nuclear receptor NR5A2 (required for follicle ovulation) are markedly reduced in PCOS ovaries (Jansen 2004). In addition, the expression of CYP19A1, the rate-limiting enzyme in estrogen synthesis, is also reduced both in conditional knockout mice and cystic human follicles (MAGOFFIN 2006).

 

 


Mice Models of Defective Ovulation


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Figure 6 | Schematic overview of human ovary showing various stages of follicle development (Cornell University 2012).

Ovulation involves a sequence of intricate events controlled by a number of signalling pathways initiated by the LH surge. Ovulation begins in mural granulosa cells and leads to cumulus cell expansion, resumption of meiosis, follicular wall rupture and luteinisation of the remaining cells in the ruptured follicle (Edson et al. 2009).

Numerous genes have been identified that influence different phases of ovulation (Matzuk 2000). Receptor interacting protein 140 (RIP140) is a co-regulator (mostly co-repressor (Cavaillès et al. 1995; Christian et al. 2004)) of the ligand-dependent family of nuclear receptors. The elimination of RIP140 in female mice results in complete infertility due to ovulatory failure. Such that following the LH surge, RIP140 knockout (RIPKO) mice are incapable of releasing oocytes from mature follicles and thus fail to ovulate; however, luteinisation occurs regardless of this failure (White et al. 2000); this continuation of luteinisation despite failure in ovulation, is phenotypically very similar to that of luteinised unruptured follicle syndrome, seen in many infertile women. This suggests that nuclear receptor suppression is as important as its activation is for correct ovarian function, and hints at the indispensible role of RIP140 in oocyte release.

Experiments designed to stimulate oocyte release through administration of gonadotrophins in RIPKO mice, were unsuccessful in initiating ovulation (White et al. 2000), but they were successful in demonstrating that the ovary itself is the culprit in the RIPKO phenotype. Later ovarian-transplantation experiment in mice were key in reinforcing the notion that this defect is independent of the HPG axis and that it lies at an ovarian level (Leonardsson et al. 2002). In addition, the ovarian transplantation experiments in RIPKO and wild type mice showed that the expression of RIP140 in other tissues is not required for ovulation, and RIP140 role in regulating ovulation lies only in the ovary (Leonardsson et al. 2002).

Folliculogenesis in RIP140 null mice is normal, but follicles are anovulatory because cumulus-oocyte-complexes (COCs) fail to expand, rupture and release the oocyte (Leonardsson et al. 2002). This expansion of COCs involves the expression of numerous matrix, neuronal and immune cell related genes (Liu et al. 2008). The ovary of the RIP140 null mouse, displays defective expression of many of these genes involved in COC expansion, an observation inline with the data from gene expression profiling of RIP140, showing its impact on expression of genes involved in extracellular matrix formation, signalling, cell-cell attachment and adhesion (Tullet et al. 2005).

The above data prompted the investigation of RIP140 downstream targets in mice, and it was revealed that in the ovary the absence of RIP140, leads to down regulation of certain members of the epidermal growth factor (EGF) network, namely amphiregulin (AREG) and epiregulin (EREG). These factors are present in the ovarian follicle and are important for oocyte maturation and cumulus expansion (Park et al. 2004). Thus, the lack of RIP140 expression in the ovary is associated with dampened EGF mediated signalling. In addition, cumulus expansion in the RIP140 null mice can be achieved by treatment with hCG and AREG, although ovulation is not rescued, suggesting the involvement of various other RIP140 targets involved in ovulation.

The combination of early and recent studies on the role of RIP140 in reproduction, suggest that RIP140 is important both as a co-activator and a co-repressor of certain genes in the ovary, although the mechanism by which RIP140 stimulates AREG is not fully elucidated, it is postulated that RIP140 may act as a transcriptional coactivator of CREB/c family members to stimulate Areg gene promoter (Park et al. 2004).

Ultimately, these finding demonstrate that ligand dependent repression of a nuclear receptor is as important a process as is its transcriptional activation, and such factors play a fundamental role in ovarian function and female infertility.


Conclusion


Transgenic mouse models have been instrumental to our understanding of the molecular components of endocrine pathways, where they’ve enabled us to not only determine the direct/indirect effects of their aberrant functioning in infertility, but to also appreciate the compensatory results of their actions in vivo.

The production of hypomorphic and null allele mice using targeted knockout approaches have allowed us to replicate deficiency and resistance syndromes seen in humans. Meanwhile, transgenic overexpression could help mimic gain of function mutations and hyper-secretion syndrome in patients, in some cases these replications in mice closely phenocopy human disorders, this could provide useful as proof of principle models and provide targets for testing potential treatments in human infertility.

However, in such experiments, not all phenotypes are anticipated, such that unpredicted phenotypes may hint at an unknown endocrine relationship, which as a result can provide us clues to previously unexplainable clinical observations. Nevertheless, genetically modified mice retain great potential in revealing many more pathways involved in human infertility.


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