Characterization of the HPG Axis with a Deficiency of FGFR1

By Rebecca Bolen Integrative Physiology, University of Colorado at Boulder

Defense Date: April 2nd, 2019

Thesis Advisor: Pei-San Tsai, Integrative Physiology

Defense Committee: Pei-San Tsai, Integrative Physiology

Alena Grabowski, Integrative Physiology Christopher Lowry, Integrative Physiology

Abstract:

Gonadotropin-releasing hormone (GnRH) is an indispensable hormone for the

commencement and maintenance of vertebrate reproduction. The development of GnRH

neurons depends greatly on fibroblast growth factor (FGF) signaling. Inactivating mutations on

FGF receptor 1 (FGFR1) have been shown to reduce GnRH, leading to compromised fertility in

humans. However, how FGFR1 deficiency impacts the structural integrity of the GnRH neuronal

network, gonadal function, and growth of the reproductive tract in adults was not well

understood. This study investigates the organization of GnRH neurons, ovarian structures, and

uterine mass in adult female mice globally deficient in FGFR1 (FGFR1-floxed mice). We

hypothesized that there would be decreased GnRH neuron numbers associated with altered

ovarian structure and uterine growth in FGFR1-deficient mice. This investigation was

accomplished by quantifying (1) GnRH neurons in different brain regions, (2) ovarian follicles

and corpora lutea, and (3) mass of the uterus, an estrogen- and progesterone-dependent organ.

Our results confirmed that there was a 66% reduction in the number of GnRH neurons in

FGFR1-floxed mice. Specifically, this reduction occurs uniformly in all brain regions where

GnRH neurons reside. This reduction in GnRH neurons was coupled with decreased numbers

of maturing or mature ovarian follicles. Surprisingly, the uterine mass of the FGFR1-floxed mice

was increased by about 40% due to unknown causes. These results suggest FGFR1 deficiency

negatively impacts GnRH neurons in all brain regions and significantly alters the ovarian and

uterine function in females. This study highlights how a single deletion of a signaling gene can

lead to downstream defects that cause infertility and other reproductive disorders.

Introduction:

The hypothalamic-pituitary-gonadal (HPG) axis is a highly organized and hierarchical

system consisting of three endocrine organs responsible for activating and maintaining

vertebrate reproduction. It begins in the hypothalamus, which releases gonadotropin-releasing

hormone (GnRH). GnRH then travels to the anterior pituitary and stimulates the release of

gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH

travel through the blood to the gonads (ovaries or testes) to stimulate gonadal maturation,

gametogenesis, and the release of estrogens and androgens. The proper function and

communication among the components of the HPG axis are crucial for many aspects of

reproduction, including puberty and fertility.

The proper functioning of the HPG axis is dependent, in part, on the binding of fibroblast

growth factors (FGFs) to fibroblast growth factor receptors (FGFRs). The FGF system consists

of 22 ligands and 4 receptors. Each receptor is an evolutionarily conserved tyrosine kinase

receptor with extracellular, transmembrane, and intracellular domains. When a FGF ligand

binds, the receptor dimerizes, resulting in the cross-phosphorylation of the intracellular domain

to initiate a myriad of intracellular signaling events (Ornitz et al., 2015). Humans with inactivating

mutations on several FGF ligands and receptors exhibited symptoms of hypogonadotropic

hypogonadism (HH), a condition of low gonadotropins, low gonadal steroids, and difficult

pubertal transition (Miraoui et al., 2013; Falardeau et al., 2008; Dodé et al., 2003).

Animal studies suggest FGF signaling likely supports HPG axis functions through

multiple mechanisms, but the most important one is its ability to support the development of

GnRH neurons, which are positioned at the apex of the HPG axis. FGFRs have been detected

in the olfactory placode, the birthplace of GnRH neurons, at the time of GnRH neuron fate

specification (Schwanzel-Fukuda et al., 1987). Specifically, an FGF ligand (FGF8) signaling

through an FGFR (FGFR1) has been shown to drive the very early phase of GnRH neuron

genesis (Tsai et al., 2011). After GnRH neurons are born, they are also guided by FGF signaling

to migrate through a large span of forebrain regions, beginning at the olfactory bulbs and ending

at the posterior hypothalamus. When FGF8 or FGFR1 were reduced in mutant mice, GnRH

neurons failed to emerge properly, leading to a significantly smaller GnRH neuronal population

in the brain (Chung et al., 2008). That being said, it is important to determine if the reduced

GnRH neurons in these mutant mice are region-specific, to deduce which developmental stage

is affected the most, and if this reduction leads to altered gonadal structure and reproductive

tract growth. At present, no study has examined downstream of the GnRH system to

understand the deficits in the gonad and reproductive tract resulting from FGFR1 deficiency.

Using Cre-loxP technology, we generated a mouse globally deficient in FGFR1, a condition

similar to humans with inactivating FGFR1 mutations (Dodé et al., 2003). The objectives of this

investigation were to examine if global FGFR1 deficiency impacts: (1) GnRH neurons in different

brain regions, (2) follicles and corpora lutea in the adult ovaries, and (3) the mass of the uterus,

an estrogen/progesterone-dependent organ. These were parameters that could not be

adequately evaluated in humans harboring FGFR1 deficiency. This study has tremendous

clinical implications since humans harboring inactivating mutations on the FGFR1 gene can

develop multiple reproductive issues including absent puberty, delayed puberty, and

hypothalamic amenorrhea (Fraietta et al., 2013).

Based on the well-established function of the HPG axis, I hypothesize that there will be

decreased GnRH neuron number associated with altered gonadal function and reproductive

tract growth in FGFR1-deficient mice. This hypothesis is based on the previous report that

FGFR1 hypomorphy leads to reduced total GnRH neurons in mice (Chung et al., 2008; Chung

et al., 2010). Consequently, a lack of GnRH should lead to a decrease in gonadotropins and

ultimately abnormal follicular development and steroid production. The latter can then lead to

the altered growth of the reproductive tract. This study aids in the understanding of how

inactivating mutations on a single signaling gene can lead to human infertility.

Material and Methods:

Transgenic Animals

The Cre/LoxP technology was used to globally delete one functional FGFR1 allele. For

this, a female mouse with a Sox2 promoter (a ubiquitous promoter expressed in the epiblast)

driving the Cre recombinase (Sox2-Cre+/-) (Hayashi et al., 2002) was bred with a male mouse

with loxP sites flanking Exon 4 of the FGFR1 gene (Fgfr1Ex4flox/-) (Hoch et al., 2006) to produce

progeny with mixed genotypes, including the experimental genotype with the global deletion of

one FGFR1 allele (abbreviated FGFR1-floxed). Due to difficulty in obtaining controls

(Sox2-Cre-/-: Fgfr1Ex4flox/-) in our breeding scheme, the paternal line of Fgfr1Ex4flox/- and their

progeny were used as the control mice. The day of birth was designated as postnatal day (PN)

0. All mice were housed in our animal facility under a 12-h light:12-h dark cycle and fed ad

libitum. At PN 20, pups were weaned and genotyped by polymerase chain reaction (PCR) of

genomic DNA isolated from tail biopsies. Only females were used for this study. All female mice

were sacrificed on diestrus around PN 60 (+/- 5 days). All animal procedures were approved by

the Institutional Animal Care and Use Committee.

Tissue Collection

Brains collected from all mice were immersion-fixed in 4% paraformaldehyde in 0.1 M

phosphate buffer at 4°C for 24 hours. They were then transferred to 30% sucrose and stored at

4°C prior to immunohistochemical (IHC) staining of GnRH neurons. Ovaries were dissected,

weighed, and immersion-fixed in Bouin’s fixative for 24 hours at room temperature, then

transferred to 70% ethanol solution and stored at room temperature. Body mass and uterine

mass were also recorded at the time of sacrifice.

GnRH IHC

The GnRH IHC followed the protocol previously published (Chung et al., 2008). Briefly,

brains were sectioned at 50-µm thickness using a cryostat and collected as floating sections into

phosphate-buffered saline (PBS). A rabbit anti-GnRH antibody (G3177) generated by our lab

was used to detect GnRH neurons. The sections were incubated for 48 hours at 4°C in G3177

(1:30,000) diluted in 0.1M PBS with 0.1% Triton X (PBST) and 4% normal donkey serum. After

a 48-hour incubation, the sections were washed with PBST and then incubated again for 2

hours at room temperature in a biotinylated donkey anti-rabbit antibody (Jackson

ImmunoResearch). Sections were washed and incubated with an avidin-biotinylated

horseradish peroxidase complex (generated and validated by our lab) for one hour at room

temperature. Then sections were washed in PBST and reacted with diaminobenzidine for color

reaction. Sections were mounted onto gelatin-coated slides, dehydrated, and coverslipped using

Permount (Fisher).

GnRH Neuronal Counts

The total number of GnRH neurons was counted through the adult forebrain at 50-μm

sections. GnRH neurons within set distances anterior and posterior to the organum vasculosum

of the lamina terminalis (OVLT) were scored for GnRH neurons by an investigator blind to the

identity of the slides. Although these set distances may exclude a few of the most anterior and

posterior GnRH neurons, this method ensured that the number of sections quantified was

consistent among all animals. Only positively stained cells that appeared neuronal and exhibited

clear nuclei were scored. For the analysis presented in Figure 4, GnRH neuron numbers were

binned into Zone 1 (anterior to OVLT; -750 to -200 μm), Zone 2 (surrounding OVLT; -150 to

+250 μm), and Zone 3 (posterior to OVLT; +300 to +1050 μm). The section at the level of the

OVLT is designated as 0 μm.

Ovarian Histology and Analysis

Ovaries were embedded in paraffin and sectioned on a rotary microtome at 12-µm

thickness. Sections were mounted onto gelatin-coated slides, deparaffinized, rehydrated, and

stained for hematoxylin and eosin (H&E). Ovaries were analyzed for the numbers of primordial

follicles, preantral follicles, antral follicles, and corpora lutea (Figure 1). Primordial follicles were

characterized as oocytes surrounded by one layer of granulosa cells with no visible space.

Preantral follicles were identified as oocytes with two or more layers of granulosa cells with

slight to no visible space. Antral follicles were recognized as those containing any antral cavity

ranging from compartments to one large continuous cavity (Griffin et al., 2006). The analysis

was performed by an investigator blind to the identity of the slides. The quantification of ovarian

structures was conducted every 5 sections, and the entire ovarian section was scored.

Figure 1. Follicular phases of maturation. Depiction of primordial (a), preantral (b), antral (c) follicles, and corpora lutea (d).

Statistics Differences in various parameters between control and FGFR1-floxed mice were

analyzed by Student’s t-test or Mann-Whitney test. All statistical analyses were performed using

Prism (GraphPad, La Jolla, CA, USA). Differences were considered significant when P < 0.05.

Results:

GnRH Neuronal Counts

To determine the effects of the FGFR1 deficiency on total GnRH neurons, the number of

GnRH neurons in the entire forebrain was counted. The Mann-Whitney test showed that in the

whole forebrain, GnRH neurons were significantly lower in FGFR1-floxed female mice

compared to their control counterparts (Figure 2). The FGFR1-floxed females showed a 66%

reduction of GnRH neurons (P < 0.05). Additionally, we examined the distribution of GnRH

neurons in reference to the OVLT, a forebrain area with the most GnRH neurons (Gill et al.,

2006). An overall analysis of GnRH neuronal distribution in reference to the OVLT is shown

(Figure 3). To quantify the region-specific impact of FGFR1 deficiency, the brains were divided

into 3 zones: Zone 1 (included sections anterior to the OVLT), Zone 2 (included sections around

the OVLT), and Zone 3 (included sections posterior to the OVLT). The Mann-Whitney test

showed that Zones 2 and 3 (Figures 4C and E) had significantly fewer GnRH neurons in the

FGFR1-floxed mice, but not the anterior Zone 1 (Figure 4A). Specifically, Zone 2 had a 55%

decrease in the FGFR1-floxed mice (P < 0.05), while Zone 3 had a 77% decrease (P < 0.05).

However, when normalized to the total number of GnRH neurons, the percentage of neurons in

FGFR1-floxed mice in each zone was not different from the controls (Figure 4B, D, and F),

suggesting GnRH neurons were uniformly reduced without a regional preference.

Figure 2. Total number of GnRH neurons in the forebrains of adult control and FGFR1-floxed female mice. N = 4 for both genotypes.

Figure 3. GnRH neuron distribution through all sections of the forebrain. Sections were analyzed in reference to the OVLT. The X-axis denotes the anterior to posterior order of the forebrain sections: Negative numbers are sections anterior to the OVLT, zero is the OVLT, and positive numbers are sections posterior to the OVLT. N = 4 for both genotypes.

Figure 4. Region-specific analysis of GnRH neurons. The GnRH distribution was separated into Zones 1, 2, and 3 according to the Materials and Methods. There were no differences in Zone 1

in total (A) and percent GnRH neurons (B). The total numbers of GnRH neurons were different between Zones 2 and 3 (C and E), but percent GnRH neurons was not different in these two zones (D and F). N = 4 for both genotypes. Mass Measurements

The evaluation of reproductive tract growth was conducted based on the mass

measurements of the body, ovaries, and uterus. The Mann-Whitney test revealed that there was

no difference in any of the mass measurements between the FGFR1-floxed mice and the

controls except the uterus (Figures 5A, C, D). In fact, the mass of FGFR1-floxed uteri was

increased by 40% compared to control uteri (P < 0.05; Figure 5D). Consistent with these

observations, the ratio of uterus mass to body mass (uterus somatic index; Figure 5E) was also

significantly increased in the FGFR1-floxed mice, but the ratio of ovarian mass to body mass

(gonadosomatic index; Figure 5B) was not different between the genotypes.

Figure 5. Female gross anatomy in control and FGFR1-floxed mice. No parameters were significantly different between the genotypes except uterine mass (D) and Uterus Somatic Index (E) (P<0.05). N = 4 for both genotypes.

Ovarian Analysis

Ovarian histological parameters were further evaluated. The ovarian structures were

characterized as primordial follicles, preantral follicles, antral follicles, and corpora lutea. This

gave us insight into (1) whether the ovaries were impacted developmentally to result in altered

primordial follicles at birth, (2) if these primordial follicles mature normally into preantral and

antral follicles, and (3) if the formation of corpora lutea (correlated with ovulation and

preparedness for pregnancy) occurred normally. The Mann-Whitney test showed that

FGFR1-floxed females had significantly reduced preantral and antral follicles (Figures 6B, C; P

< 0.05), but no difference in primordial follicles and corpora lutea (Figures 6A, D).

Figure 6. Assessment of ovarian structures. Ovarian structures were classified as primordial (A), preantral (B), antral (C) follicles, and corpora lutea (D). There was a significant decrease in preantral and antral follicles in FGFR1-floxed females (B, C). N=4 for both genotypes.

Discussion:

There are many steps required for GnRH neurons to properly stimulate the activity of the

HPG axis. Importantly, it has been shown that FGFR1 deficiency disrupts the fate specification

of GnRH progenitor cells. This disruption leads to a decrease in the developed GnRH neuronal

population at birth (Chung et al., 2008; Chung et al., 2010). Based on these findings, the current

study aimed to characterize the female HPG axis post-puberty (around PN60). Specifically, we

examined the GnRH system and further down the axis at the level of the ovaries to understand

the downstream effects of an FGFR1 deficiency. Towards this goal, GnRH neuronal count was

obtained and gonadal histology was analyzed.

We discovered that GnRH neuron number in the forebrain of FGFR1-floxed mice was

significantly reduced compared to their control counterparts. This was consistent with the

previous findings that FGFR1 deficiency impaired the genesis of GnRH neurons (Chung et al.,

2008; Chung et al., 2010) and suggested that this loss of GnRH neurons is likely irreversible.

When dividing the neurons into zones, we found that there was a significant decrease in GnRH

neuronal numbers in Zones 2 (around the OVLT) and 3 (posterior to the OVLT) (Figures 4C, E).

This was initially of interest due to the developmental history of GnRH neurons. For example,

the emergence of GnRH neurons in the olfactory placode lasted approximately 1.5-2 days of

fetal development. The early-emerging GnRH neurons migrated the farthest and settled in the

most posterior forebrain region, whereas GnRH neurons that emerged later migrated to the

most anterior forebrain (Wu et al., 1997). A reduction in the number of neurons in Zones 2 and 3

initially suggested that the development and migration of GnRH neurons that had emerged

earlier in development may be compromised. However, this regional difference disappeared

after normalizing GnRH neurons in each zone to total GnRH neurons (Figures 4D, F),

suggesting GnRH neurons were uniformly decreased in all brain regions of the FGFR1-floxed

mice. In other words, FGFR1-floxed mice had a smaller population of GnRH neurons to begin

with, and the reduced population migrated normally to their final destinations.

The gross anatomy of the mice was analyzed to give insight into the growth of the

ovaries and female reproductive tract. None of the parameters were significantly different

between controls and FGFR1-floxed females besides the uterus mass. The reason for the

increased uterus mass in FGFR1-floxed females is currently unclear. Uterine growth is

stimulated by ovarian steroids (estrogens and progesterone). FGFR1-foxed mice exhibited

reduced GnRH neurons, suggesting low gonadotropins and consequently low ovarian steroids.

In this case, there should be a decrease, instead of an increase, in uterus mass. One possibility

is that the uterus in FGFR1-floxed females gained mass not by conventional tissue growth, but

by abnormal fluid accumulation when FGFR1 signaling is locally absent in the uterus.

Unfortunately, we cannot determine the exact cause without hormonal analysis of

gonadotropins, ovarian steroids, and uterine histology. The increase in uterus mass is clearly a

phenomenon requiring further investigation.

The ovarian histology was analyzed based on the number of different follicle types and

number of corpora lutea. There was a much higher count of corpora lutea than any other follicle

types, an observation previously reported for female mice sacrificed on diestrus (Caligioni,

2009; Griffin et al., 2006). There was no difference in this number between the control and

FGFR1-floxed mice. Since corpora lutea are a sign of ovulated follicles, this suggests there was

a normal level of ovulation, a function controlled by the gonadotropin LH. Similarly, there was no

difference in the number of primordial follicles between the genotypes, indicating that each

animal began with approximately the same number of primordial oocytes at birth. The

differences seen between the genotypes were displayed in the preantral and antral follicles

(Figures 6B, C). Both follicle types are in the process of maturation to become ovulation-ready.

A reduction in both preantral and antral follicles suggests a complication in the follicle

maturation process. This could be due to a decrease in FSH levels, as the primary function of

FSH is to stimulate the growth and development of follicles (Méduri et al., 2002). Since FSH is

under the control of GnRH, it stands to reason that reduced GnRH neurons lead to reduced

FSH, which consequently reduced follicular maturation.

Future studies should analyze serum and pituitary gonadotropins, as well as sex

steroids, to better describe the differences seen in the ovarian histology and uterine mass

measurement. This will provide insight into whether there are sufficient levels of gonadotropins

being stored in the pituitary and released into the bloodstream in order to stimulate

gametogenesis. If not, this would illustrate why there were fewer mature follicles in

FGFR1-floxed mice. Overall, this study has demonstrated that mice harboring an FGFR1

deficiency exhibited multiple defects along the HPG axis, including reduced GnRH neurons,

abnormal uterine mass, and reduced ovarian follicular maturation. These data provide a

foundation for understanding how inactivation mutations on FGFR1 can lead to severe

reproductive disorders, such as HH, in humans.

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