Biology bachelor thesis
Regulation of spermatogenesis
by estrogen in zebrafish
Jessie Poelsma
Supervisors: Luiz Henrique de Castro Assis
Rüdiger W. Schulz
Utrecht University, Developmental Biology
23-1-2015
Source: Lynn Ketchum
Table of contents
Summary ................................................................................................................................................. 3
Chapter 1 – Spermatogenesis in fish ....................................................................................................... 4
1.1
Testicular structure ..................................................................................................................... 4
1.2
Spermatogenetic process ............................................................................................................ 4
1.2.1 From spermatogonia to spermatozoa ........................................................................................ 5
1.2.1.1
Mitotic phase ................................................................................................................... 5
1.2.1.2
Meiotic phase .................................................................................................................. 6
1.2.1.3 Spermiogenic phase ............................................................................................................ 6
1.2.2 Somatic cells ............................................................................................................................... 7
1.2.2.1 Leydig cells........................................................................................................................... 7
1.2.2.2 Sertoli cells .......................................................................................................................... 7
1.2.2.3 Myoid cells........................................................................................................................... 8
1.2.2.4 Capillaries ............................................................................................................................ 8
Chapter 2 – Hypothalamic-pituitary-gonadal axis................................................................................... 9
2.1 From hypothalamus to pituitary ....................................................................................................... 9
2.2 From pituitary to gonads ................................................................................................................... 9
2.3 Effects of Fsh/Lh in the testes ......................................................................................................... 10
2.4 From gonads to brain ...................................................................................................................... 11
2.5 Autocrine/paracrine regulatory loops ............................................................................................. 11
Chapter 3 – Sex steroids: androgens and estrogens ............................................................................. 12
3.1 Production ....................................................................................................................................... 12
3.2 Effects .............................................................................................................................................. 13
3.2.1 Germ cells ................................................................................................................................. 13
3.2.2 Sertoli cells ............................................................................................................................... 13
3.2.3 Leydig cells................................................................................................................................ 14
3.2.4 Myoid cells................................................................................................................................ 14
1
Chapter 4 – Current research by the Developmental Biology group at Utrecht University ................. 15
Chapter 5 – Relevance ........................................................................................................................... 17
5.1 Fundamental research..................................................................................................................... 17
5.2 Ecotoxicology................................................................................................................................... 17
5.3 Breeding .......................................................................................................................................... 18
Chapter 6 – Research on the influence of estrogen on zebrafish testes .............................................. 20
6.1 Introduction ..................................................................................................................................... 20
6.2 Material and methods ..................................................................................................................... 20
6.3 Results ............................................................................................................................................. 21
6.4 Discussion and conclusion ............................................................................................................... 22
2
Summary
The aim of this thesis is to give an insight into how hormones in general, and estrogen
specifically, regulate spermatogenesis in zebrafish.
First, information on the spermatogenic process, the somatic and germinal cell types involved
in this process and the main hormonal substances that regulate spermatogenesis, is reviewed. This
information can be found in chapters one, two and three.
Then, the research that is currently carried out at the Developmental Biology group at Utrecht
University is introduced in chapter four. The questions, or problems, that are still open for
investigation are presented.
The information and research on the topic of hormonal control of spermatogenesis is not only
of fundamental interest, but also has an applied background, which will be discussed as well. Chapter
five presents how this research may contribute to gain knowledge that may yield a healthier
environment and maybe even an improved economy.
In the concluding chapter six, the experiment that was carried out during my internship is
described. This experiment was performed with the objective to clarify if the estrogen effects on
spermatogenesis in zebrafish in vivo are direct effects on the testis or indirect effects via feedback
mechanisms on the brain and pituitary level. This was done by incubating testis tissue with estrogen
in vitro using a primary tissue culture system, in order to compare the results with those obtained in
vivo.
3
Part 1: Review
Chapter 1 – Spermatogenesis in fish
1.1 Testicular structure
The testes of fish are composed of two compartments: the interstitial and the tubular
compartment. The interstitial compartment contains only somatic cells, such as Leydig cells, blood
vessels, connective tissue, macrophages and mast cells, lymphatic vessels and neural cells. The
tubular compartment is separated from the interstitial compartment by a basement membrane,
which is surrounded by myoid cells. Within this tubular compartment, the seminiferous epithelium is
found, which consists of (somatic) Sertoli cells together with their accompanying developing germ
cells (Schulz et al., 2010). The seminiferous epithelium encompasses the tubular lumen, which is
filled with fluid. In this lumen, the spermatozoa are released after completing their developmental
process.
1.2 Spermatogenetic process
Spermatogenesis is a complex and coordinated process in which male germ cells transform
from single diploid spermatogonia into small, haploid and highly specialized spermatozoa (de Waal,
2009). These spermatozoa have the capacity to fertilize the female eggs.
Spermatogenesis in fish is comparable to mammalian spermatogenesis. The most
characteristic difference between mammalian and fish spermatogenesis, is the organization of the
seminiferous epithelium. The seminiferous epithelium of mammals is organized in stages (non-cystic
spermatogenesis), whereas in the seminiferous epithelium of fish, the Sertoli cells and germ cells are
organized in well-defined
structures called cysts
(cystic spermatogenesis).
In the cystic form of
spermatogenesis, depicted
in
figure
1.1,
the
developmental path starts
with one or two Sertoli
cells cradling one single
undifferentiated
spermatogonium.
When
this
spermatogonium
divides into daughter cells
and starts differentiating in
order
to
become
spermatozoa, the Sertoli
cells that surround this
clone also proliferate in
order to maintain the Figure 1.1 – Cystic spermatogenesis in fish. Depicted are cysts of Sertoli cells (SE), with consecutive stages of
cystic form and support the germinal cells: type A spermatogonia in different stages, type B spermatogonia, different phases of meiotic
spermatocytes (Z/L, P, D/MI, S/MII), spermatids (E1, E2, E3), spermatozoa (SZ). Also myoid cells (MY), Leydig cells
increasing number of germ (LE) and capillaries (BV) are specified. Source: (Schulz et al., 2010)
4
cells within the cyst. The cysts, each containing germ cells at one and the same developmental stage,
are positioned next to each other along the basement membrane. When the germ cells have gone
through all the developmental stages, and have become spermatozoa, these spermatozoa are
released into the lumen of the seminiferous tubule.
Sertoli cells concerned with non-cystic spermatogenesis have to produce many different
products for their surrounding germ cells, since these associated germ cells are in a variety of
different developmental stages. In cystic spermatogenesis, however, the Sertoli cells are in contact
with a fewer variety of developmental stages of germ cells. Therefore, the Sertoli cells do not need to
produce as many different products for their associated germ cells. This causes the Sertoli cells
concerned with cystic spermatogenesis (as found in fish) to be highly efficient, compared with Sertoli
cells concerned with non-cystic spermatogenesis (as found in mammals).
1.2.1 From spermatogonia to spermatozoa
Based on morphology and physiology, spermatogenesis can be divided into three phases: a
mitotic phase, a meiotic phase and a spermiogenic phase. During the mitotic phase the germ cell
number increases due to a species-specific number of mitotic spermatogonial divisions. In the
meiotic phase recombination and reduction of DNA takes place, yielding haploid cells. The haploid
germ cells transform into spermatozoa during the spermiogenic phase (de Waal, 2009; Schulz et al.,
2010).
1.2.1.1 Mitotic phase
A spermatogonial stem cell can go through two distinct types of division. Which cells will go
through which division, is determined by the somatic environment with signaling factors (Schulz et
al., 2010).
One of the division types is stem cell self-renewal: a division that gives rise to new stem cells
and guarantees prolonged fertility. The other division type gives rise to two daughter cells, which are
predetermined to go through the process of differentiation into spermatozoa. The daughter cells
remain interconnected by a cytoplasmic bridge (de Rooij & Grootegoed, 1998). In this way, all
processes in the daughter cells can be performed simultaneously.
Spermatogonia can be categorized into A and B type spermatogonia, based on the amount of
heterochromatin in their nuclei, nuclear shape, nucleolar characteristics and the amount of cells in a
cyst (Leal, Cardoso, et al., 2009). Type A spermatogonia lack heterochromatin, whereas type B
spermatogonia have high amounts of heterochromatin in their nuclei. Type A spermatogonia can be
further subcategorized into type A differentiated (Adiff) and type A undifferentiated (Aund)
spermatogonia. In zebrafish, within the group of undifferentiated, single spermatogonia, Aund and
Aund* spermatogonia can be distinguished (de Waal, 2009). The Aund* spermatogonia are thought to
make up the stem cell pool, however, Aund are seen to have the capacity to behave as stem cells as
well. This nomenclature is only used within zebrafish, since these subdivision of spermatogonia is
species-specific and thus may differ from other fish species.
After the type Aund spermatogonia have divided into two daughter cells, these spermatogonia
form the first stage of clones that remain interconnected via the cytoplasmic bridge. These
spermatogonia undergo their first differentiation into Adiff spermatogonia. Following more mitotic
divisions of these daughter cells, chains of daughter spermatogonia arise. Eventually, the clones will
transform into type B spermatogonia. Type B spermatogonia can be classified as B1, B2, B3, etcetera.
However, this distinction is hard to make based only on morphology. Therefore, B spermatogonia are
usually grouped into early and late B spermatogonia, based on heterochromatin structure and the
5
number of germ cells inside the cyst. The number of type B spermatogonial generations is species
specific. The cells that are in the final B spermatogonial stage, undergo a last mitotic division to give
rise to primary spermatocytes (Leal, Cardoso, et al., 2009).
Since this nomenclature can – for parts or as a whole – be applied to other fish species as well,
and is parallel to the mammalian nomenclature, these distinguishable spermatogonial stages can be
used in comparative studies of spermatogenic regulation. Moreover, a nomenclature with clear
characteristics of the different developmental stages makes identification of these individual stages
easier. When these stages are recognized in an easier manner, cysts with germ cells in particular
developmental stages can be used in further investigations to, for example, try to identify the
substances needed by the germ cells at these particular stages, and thus gain insight in the molecular
pathways that are activated in Sertoli cells, Leydig cells, and/or germ cells per developmental stage.
1.2.1.2 Meiotic phase
The primary spermatocytes that have been formed during the mitotic phase undergo the first
meiotic division. Within this process, several stages can be distinguished on the basis of
chromosomal condensation (de Waal, 2009). During the first meiotic division, recombination of DNA
takes place, and the chromosomes separate and get distributed over two daughter cells, which are
now called secondary spermatocytes. These secondary spermatocytes are ‘2n’ instead of 2n, because
they are not identical considering their DNA.
The secondary spermatocytes are very short lived; they quickly enter the second meiotic
division (Leal, Cardoso, et al., 2009; Schulz et al., 2010). During the second meiotic division, the
chromatids separate and get distributed over two daughter cells: the spermatids. So after the meiotic
phase, one primary spermatocyte (4n) has given rise to four haploid (1n) spermatids.
1.2.1.3 Spermiogenic phase
Directly after passing through the meiotic phase, the germ cells develop into spermatids which
have small and dense nuclei. Spermiogenesis is the process during which spermatids transform into
spermatozoa. Within this process, several distinctive morphological changes take place (Miura &
Miura, 2004), such as formation of a sperm head containing DNA in a condensed nucleus,
construction of a mid-piece containing mitochondria, formation of a flagellum for propulsion and
transformation of the Golgi-apparatus into an acrosome (at least in teleost fish; spermatozoa of
other fish species may lack an acrosome or differ in other cellular characteristics).
In zebrafish, three types of spermatids can be discerned: early spermatids (E1), intermediate
spermatids (E2), and final spermatids (E3). This classification can be made on the basis of nuclear
condensation. There are also three forms of spermiogenesis (I, II and III) based on nuclear rotation
and the position of the flagellum to the nucleus (Schulz et al., 2010).
Lastly, the spermatozoa are released into the lumen of the seminiferous tubules by a process
called spermiation, where the spermatozoa detach from their Sertoli cells (Schulz et al., 2010).
During sperm maturation, which happens after the spermatozoa are released into the lumen,
the spermatozoa get their full functionality, that is, they become motile and capable of fertilization.
This process only consists of physiological changes (Miura & Miura, 2004); the morphological changes
have been completed in the previous stages.
6
1.2.2 Somatic cells
1.2.2.1 Leydig cells
One of the functions of Leydig cells is to produce androgens, amongst other functions. They
are located in the interstitial compartment between the seminiferous tubules (Silverthorn, 2010),
and tend to be clustered near the capillaries (Neill & Knobil, 2006) in ring-like structures (Leal,
Cardoso, et al., 2009). In the zebrafish embryo, the Leydig cells are inactive considering the
production of androgens, since the gonads develop only after about two weeks after the embryos
hatch. After two weeks, production of androgens is necessary to develop the male characteristics
and achieve fertility.
1.2.2.2 Sertoli cells
Sertoli cells are the only somatic cells found in the seminiferous epithelium (de Rooij &
Grootegoed, 1998; Miura & Miura, 2004). They support the maturing germ cells by secreting the
growth factors and differentiation factors spermatogonia need during their development towards
spermatozoa. Moreover, they coordinate spermatogenic events, through which the seminiferous
epithelium gets its well-organized function and structure. They also secrete the fluid that fills up the
tubular lumen (Schulz et al., 2010).
Sertoli cells have receptors for Follicle Stimulating Hormone (Fsh) and testosterone, which
causes these hormones to be the main regulating substances of spermatogenesis. They control the
maturation and supporting activities of Sertoli cells. Sertoli cells secrete different products at
different times. In this way they determine when their associated germ cells will undergo the next
developmental stage (de Franca et al., 2015).
Depending on the developmental stage of their associated germ cells, neighboring Sertoli cells
in the seminiferous tubules can be connected to each other via tight junctions. These tight junctions
form the blood-testis barrier, which, once this barrier is established, makes it impossible for most
molecules to enter the lumen of the seminiferous tubules (Silverthorn, 2010). In this way, the bloodtestis barrier prevents inflammation of the testes, by preventing cells of the immune system to reach
the spermatozoa and recognize them as foreign bodies. Thus, the only cells that can provide
immunological defense within the lumen of the seminiferous tubules are the Sertoli cells. Next to
that, they phagocytize apoptotic germ cells, residual sperm and bodies discarded by spermatids.
Moreover, the blood-testis barrier makes it possible for a separate micro-environment to arise within
the tubular compartment, wherein post-meiotic cells will find exactly what they need for their
development and proper functioning (Schulz et al., 2010).
A remarkable finding has been done by Leal, Cardoso, et al. (2009), who found that in
zebrafish, the tight junctions of Sertoli cells at the basal part of the germinal epithelium occur in cysts
with early spermatids and cysts with germ cells in further developmental stages. This shows that the
blood-testis barrier in zebrafish is build up at the end of the meiotic phase, whereas in mammals,
germ cells are already separated from the interstitial milieu at the beginning of the meiotic phase. It
thus seems that either there is no need for a protective barrier for the germ cells of zebrafish in the
meiotic phase because of different characteristics of the germ cells between mammals and zebrafish,
or that this protective barrier is needed at this point in zebrafish as well, but that other structures are
involved by setting up this barrier.
Since spermatogenesis in fish has a cystic form, the final differentiation of Sertoli cells takes
place asynchronously. This reflects that each cyst functions as an independent unit of germ cells and
Sertoli cells, which follows its own path of development. In mammals, however, the final
7
differentiation of Sertoli cells happens in a set time frame, namely puberty, wherein all Sertoli cells
go through this process simultaneously (Schulz et al., 2010).
1.2.2.3 Myoid cells
Myoid cells surround the seminiferous tubules, and are therefore in contact with both the
interstitial compartment and the tubular compartment. Their organization varies per species.
Myoid cells contain actin filaments, which gives them contractile characteristics, even though
they are not innervated (Maekawa et al., 1996). These contractions cause the testicular fluid in the
lumen, containing mature spermatozoa, to move down the seminiferous tubules.
Some of the substances myoid cells produce (extracellular matrix components and several
growth factors, among others) are known to affect the function of Sertoli cells. Myoid cells also have
androgen receptors. Both facts point at a role for myoid cells not only in maintaining the structure of
the seminiferous tubules, but also in the regulation of spermatogenesis (Maekawa et al., 1996).
1.2.2.4 Capillaries
Capillaries are situated in the interstitial compartment between the seminiferous tubules.
Endothelial cells of the capillaries produce a unique protein constituent of tight junctions. Another
such protein that is produced by endothelial cells, is also produced by Sertoli cells and myoid cells.
These facts suggest that endothelial cells have a function in maintaining the blood-testis barrier. It
has also been suggested that they mediate the response to Luteinizing Hormone (Lh) of the Leydig
cells (Neill & Knobil, 2006). The primary function of endothelial cells, however, is of course the
regulation of filtration and resorption of fluid and proteins.
8
Chapter 2 – Hypothalamic-pituitarygonadal axis
The hypothalamic-pituitary-gonadal (HPG) axis is the main regulator of reproduction in
vertebrates. The HPG-axis of fish resembles the HPG-axis of higher vertebrates in a remarkable way.
All major components and functions found in the mammalian HPG-axis are conserved in the fish
HPG-axis (Golan, Biran, & Levavi-Sivan, 2014).
2.1 From hypothalamus to pituitary
The reproductive pathway starts with the secretion of peptide hormones from the
hypothalamus: Gonadotropin Releasing Hormone (Gnrh) and Gonadotropin Inhibiting Hormone
(Gnih). These hormones control the secretion of gonadotropins from the anterior pituitary
(Silverthorn, 2010). In fish, dopamine also plays a pivotal role in inhibiting gonadotropin secretion.
Gnrh is a decapeptide produced in hypothalamic neurons. Once these neurons receive neural
input, Gnrh is produced and stored in the axons of these neurons, at the nodes where the axon splits
to make contact with dendrites from other Gnrh-neurons (Gothilf et al., 1996). Gnrh regulates the
endocrine output of gonadotropins (such as Fsh and Lh) by the anterior pituitary via a G-protein
coupled receptor. Gnrh stimulates the expression of gonadotropin receptors, and stimulates
secretion of gonadotropins. Therefore Gnrh can be seen as an interface between the neural and the
endocrine system.
For relatively long it was thought that the only way in which secretion of gonadotropins could
be inhibited, was a decrease in the secretion of Gnrh. However, quite recently it was found that there
was a Gonadotropin Inhibiting Hormone (Gnih), which has – as the name already implies - an
inhibiting function on the release of gonadotropins by the pituitary. It has been proven that GnIH is
capable of decreasing plasma LH levels and inhibiting gonadotropin biosynthesis in vitro (Tsutsui,
2005). GnIH has its own G-protein coupled receptor as a target, but it can also decrease the activity
of GnRH-receptors (Ubuka et al., 2013). These results have been found in mice, birds and hamsters;
therefore, there is a possibility that these findings do not hold for fish, but since the endocrine
control mechanisms are very well preserved throughout the evolution, it is possible that Gnih in fish
controls gonadotropin release in a similar way.
Next to Gnrh and Gnih, dopamine also influences the production and secretion of
gonadotropins in fish. A tonic dopaminergic inhibition is exerted on the release of gonadotropins. It
acts directly on the gonadotropins by inhibiting spontaneous gonadotropin release, and it also
suppresses the stimulatory effect of Gnrh (Lin et al., 1989).
2.2 From pituitary to gonads
In fish, GtH-I and GtH-II are dimeric glycoproteins. They consist of an α-subunit, which is
common, and a β-subunit, which is different per species. This GtH-I and GtH-II are the equivalents of
mammalian gonadotropins FSH and LH, respectively. While FSH and LH are produced by the same
cells of the mammalian pituitary, GtH-I and GtH-II are produced in different cells of the teleost
pituitary. Gonadotropins are stored in the pituitary (Evans & Claiborne, 2005). The names GtH-I and
9
GtH-II are old names for the fish gonadotropins. Later in time, the names for the fish equivalents
have been changed into Fsh and Lh to indicate similarity to mammalian FSH and LH.
FSH and LH affect target cells via their own designated G-protein coupled receptors. The roles
of Fsh and Lh however, are not as strictly separated in fish as the roles of FSH and LH in mammals.
This follows from promiscuity in ligand specificity and in overlapping expression sites of the Fsh and
Lh receptors in the testes (de Waal, 2009). However, it appears that a preference of the receptors for
their ‘corresponding’ gonadotropin exists. Moreover, the amount of the ‘non-corresponding’
gonadotropin needed to cross-activate the receptor may exceed natural plasma concentration levels
(Schulz et al., 2010). Therefore, Lh- and Fsh-receptors in fish may also be considered as
physiologically specific, even though this specificity is not as strict as in mammals.
Fsh and Lh control the secretion of steroid sex hormones produced by the gonads. In addition,
Fsh stimulates Sertoli cells to produce inhibin and the molecules needed for spermatogenesis
(Johnson & Gomez, 1977). Next to the path gonadotropins follow from the pituitary to the gonads,
the gonadotropins serve in a short loop feedback to the hypothalamus, inhibiting the release of
Gnrh.
2.3 Effects of Fsh/Lh in the testes
Fsh stimulates the gonads in a direct manner. It directly stimulates Sertoli cells, which have a
function in nourishing of and caring for the maturing germ cells. Fsh binds to a receptor on the Sertoli
cell membrane, through which adenylyl cyclase is activated. This second messenger causes the
Sertoli cell to produce growth factors and other cell products, which are secreted in a paracrine
manner towards the spermatogonia. The spermatogonia need these Sertoli cell products to increase
proliferation and decrease apoptosis. Next to regulating these specific spermatogenic processes, the
Sertoli cell products also allow spermatogenesis in general to take place (Rhoades & Bell, 2009).
Lh directly stimulates endocrine cells - the Leydig cells - which have a function in the
production of 11-ketotestosterone (11-KT) and testosterone (T) in zebrafish. 11-KT and T have a
stimulating effect on the Sertoli cells, which enables these cells to construct the cell products the
maturing spermatogonia need. In this way, Lh has an indirect effect on Sertoli cells and thus an
indirect effect on the increase of proliferation and decrease of apoptosis of the developing germ
cells.
Next to Lh-receptors, Leydig cells also express Fsh-receptors. In order for Fsh to be able to
influence the expression of several genes, steroid production is required. This shows that this action
of Fsh is mediated by an indirect pathway. However, the majority of genes in the Leydig cells are
directly influenced by Fsh in their gene expression, without the prerequisite of steroid production,
and these genes are highly relevant for the onset of spermatogenesis (Sambroni, Lareyre, & Le Gac,
2013). Fsh also has a strong steroidogenic effect.
There is no effect of Fsh and Lh directly on the germ cells, since they have no receptor for
these hormones. Both Fsh and Lh thus stimulate the production of gametes only via Sertoli cells and
Leydig cells.
10
2.4 From gonads to brain
Steroids stimulate production of Gnrh whilst inhibiting their release. Steroids do not have a
direct effect, but influence Gnrh production and secretion via several indirect processes, such as
aromatization (Gupta, 2011). In this way, the stocks that initially have been used to send a signal
from the hypothalamus to the pituitary, are replenished. This is a long loop feedback from the
gonads to the hypothalamus. Next to the influence steroids have on Gnrh, they also influence the
tonic dopaminergic inhibitory tone by increasing the dopamine turnover rate. By increasing the
responsiveness of the pituitary to Gnrh, it is ensured that Lh plasma concentrations do not change
(Trudeau et al., 1993). Several studies showed estrogens cause harm to fertility in teleost fish on all
levels of the HPG-axis (Filby, Thorpe, & Tyler, 2006; Zhang et al., 2008).
There is also a short loop feedback from the gonads to the pituitary. The steroid hormones
produced by the gonads directly regulate the gonadotropin release, which is dose-dependent. When
there is a low dose of steroid hormones there is an absence of negative feedback, through which the
gonadotropin level increases. With moderate or high doses of steroid hormones, the negative
feedback is generated and the gonadotropin level decreases (Silverthorn, 2010).
Furthermore, Sertoli cells in the gonads produce and secrete inhibin and activin. These
substances are part of de Transforming Growth Factor- (Tgf) family. Inhibins inhibit Fsh secretion
and activin stimulates Fsh secretion by the pituitary.
2.5 Autocrine/paracrine regulatory loops
The activin and inhibin produced in the gonads not only have a function in the regulation of
the gonadotropin release from the pituitary (Silverthorn, 2010). They also serve as hormones in an
autocrine and paracrine regulatory loop. Inhibin improves steroidogenesis in the Leydig cells via an
increased expression of enzymes and the Lh-receptor. Activin promotes spermatogenesis: it
stimulates the proliferation and differentiation of spermatogonia (Rocha, Arukwe & Kapoor, 2008).
Androgens themselves inhibit the production of androgens in the Leydig cells. This pathway
thus comprises an autocrine negative feedback loop (Schulz et al., 2008). Indirect estrogenic
inhibition of steroidogenesis has also been reported (de Waal et al., 2009), however, this was an in
vivo experiment, which thus only gives an indication that the effect of estrogens on spermatogenesis
is indirect, but no definite conclusion can be drawn on this yet.
It appears that in general, input on spermatogenesis by gonadotropins and sex steroids can be
fine-tuned by other pituitary hormones (i.e. growth hormone) and local signals, so this involves short
loop top-down influences as well as paracrine and autocrine regulation. The mechanisms through
which this fine-tuning mediates spermatogenesis are for most parts still unknown. It may involve
differential expression of receptors for hormones and/or growth factors in each spermatogenic cyst.
Furthermore, it seems that the hormones and/or growth factors ensure a smooth transition and
balance between the different micro-environments required for each developmental stage, in order
for germ cells to go down their developmental paths (Schulz et al., 2010).
11
Chapter 3 – Sex steroids: androgens and
estrogens
3.1 Production
Androgens and estrogens are categorized as steroid hormones, together with glucocorticoids,
mineralocorticoids and progestogens. The classification of molecules into one of these five groups of
steroid hormones is based on the kind of receptor to which they bind. Androgens work via a nuclear
receptor. The amount of different estrogen receptors differs per species. In fish, three different
estrogen receptors have been reported (Hawkins et al., 2000). Nowadays, in fish, a differentiation is
made between ER-α, ER-β1 and ER-β2 (Filby & Tyler, 2005; Menuet et al., 2002). These receptors in
fish are nuclear receptors, which act as ligand inducible transcription factors (Filby & Tyler, 2005).
Steroid hormones are built up out of four interlinked carbon rings; three 6-carbon rings
followed by one 5-carbon ring. Steroidogenesis is the biological process by which cholesterol is
converted into other steroids (Hanukoglu, 1992). Which steroid will be made in a particular cell,
depends on the regulation of expression of genes (such as cyp11a, cyp17, cyp19, 3βhsd2, and
17βhsd4 (Ma et al., 2011)) in that cell.
At first, cholesterol is produced from acetate or from cholesterol ester stores in lipid droplets
within the cell, or by uptake of
low density lipoproteins that
contain cholesterol (Bowen,
2001). After this production of
cholesterol, it gets transported
into the mitochondria under
the influence of Steroidogenic
Acute
Regulatory
Protein
(Star). Thereafter, several
steroidogenic enzymes come
into play. P450-associated
enzymes (such as hydroxylases
and lyases) convert cholesterol
into
pregnenolone.
Pregnenolone is transported
from the mitochondria into the
endoplasmatic
reticulum,
where
conversion
to
progestogens and androgens
takes place (Bowen, 2001). In
order to convert androgens
into estrogens, the enzyme
Figure 3.1 – An overview of human steroidogenesis. In other species (such as
aromatase
is
needed.
Aromatase
causes
fish) steroidogenic pathways are similar, but differences may occur in the final
hydroxylation of the 19-methyl group of androgens,
produces. Source: (Häggstrom & Richfield, 2014)
followed by elimination of this group and
12
aromatization of the A-ring (C1, 2, 3, 4, 5, 10) through which estrogens arise (see figure 3.1 for an
overview of steroidogenesis).
In zebrafish, the most important estrogen is 17-estradiol (E2), just like in most vertebrates..
Estrogens are known to inhibit androgen synthesis (de Waal, 2009). 11-KT and T are the most
important androgens in zebrafish. The difference between T and 11-KT is that 11-KT is not
aromatizable.
3.2 Effects
3.2.1 Germ cells
No receptors for androgens have been found on germ cells of fish (Schulz et al., 2010). In cases
where androgen receptors were found to be expressed in germ cells of rodents, this was not
required to reach normal fertility (Johnston et al., 2001; Tsai et al., 2006). This means that most likely
there is no effect of androgens and/or estrogens on the germ cells without interference of other
substances or pathways. However, in amphibians, with similar cystic spermatogenesis compared to
fish, estrogens are reported to promote spermatogonial proliferation (Chieffi, Amato, Staibano,
Franco, & Tramontano, 2000). In fish it has been reported that only stem cell self-renewal is
stimulated by E2. It has not been found that E2 treatments promote spermatogonial proliferation
and meiosis (Miura & Miura, 2004).
It has been shown that estrogens also disrupt spermatogenesis in adult zebrafish. They do so
in at least two ways. First estrogens cause a reduced availability of type B spermatogonia. Second,
entry into meiosis and spermiogenesis of germ cells is inhibited. These effects are not likely to be
direct, since an ex vivo study showed that meiotic and spermiogenic stages can be present when
estrogen concentrations are high, but this needs further investigation (de Waal et al., 2009). This
means that estrogens probably act as a negative feedback towards the hypothalamic-pituitarygonadal axis.
Estrogen receptors may not have been found in spermatogonia, but they have been found in
the secondary spermatocytes, spermatids and mature sperm of channel catfish (Ictalurus punctatus)
(Wu et al., 2001). Next to that, somatic cells in the zebrafish testes also express estrogen receptors.
This indicates a role for estrogens in regulating gene expression in the testes of fish. Indeed, in
several studies, estrogens have already been shown to have a direct or indirect effect on the
regulation of gene expression with a function in steroidogenesis or spermatogenesis (Schulz et al.,
2010).
3.2.2 Sertoli cells
Estrogen receptors have already been found in the Sertoli cells of zebrafish testes (Liu et al.,
2009), and also the expression of androgen receptors has been reported (de Waal, 2009).
Miura et al. (1999) reported that estrogens stimulate spermatogonial stem cell renewal
probably via Sertoli cells. The pathway by which this happens, is depicted in figure 3.2.
Androgen receptors have been found in Sertoli cells, suggesting that androgens exert
their function via the somatic cells in the testes. It seems, for example, that 11-KT is a
factor involved in the initiation of spermatogonial differentiation towards meiosis, but
there is a possibility that other factors produced by Sertoli cells, such as antiMüllerian hormone (Amh) and activin, mediate the action of 11-KT in
13
Figure 3.2 – Possible estrogen-dependent pathway of
stem cell self-renewal in fish. Source: (Schulz et al., 2010)
a stimulatory or inhibitory manner (Miura & Miura, 2001). A possible pathway is
shown in figure 3.3.
It has been shown that androgens highly influence gene expression. Several
transcription factors involved in testicular development are regulated by both T and
11-KT. Possibly, they exert substance-specific effects on gene expression (Schulz et
al., 2010).
3.2.3 Leydig cells
Zhou et al. (2002) found that estrogen receptors are expressed by Leydig
cells in mice. Therefore it is possible that Leydig cells in zebrafish also express an
estrogen receptor on which estrogens can act.
Androgen receptors also have been found in Leydig cells (de Waal,
2009), even though they produce androgens themselves. Androgens have an
inhibiting effect on the androgen production by the Leydig cells (Schulz et al.,
2008).
Figure 3.3 – Possible 11-KT-dependent
pathway of spermatogonial proliferation
towards meiosis in fish. Source: (Schulz et al.,
2010)
3.2.4 Myoid cells
The existence of androgen receptors has been reported in the myoid cells of rodents. Estrogen
receptors however, are not present in rodent myoid cells (Nakhla et al., 1984). In androgen receptor
knockout mice, the lack of androgen receptors resulted in similar fertility, but a decreased sperm
output was measured as compared to control mice (Wang et al., 2009).
The effects of androgens on myoid cells in fish however, need further investigation, since these
effects are not fully unraveled yet. Next to that, the existence or lack of estrogen receptors in fish
myoid cells has not been reported, so an effect of estrogens on myoid cells in fish can therefore not
be precluded.
14
Part 2: Research proposal
Chapter 4 – Current research by the
Developmental Biology group at
Utrecht University
As follows from the overview of information in chapters one, two and three, in vertebrates the
endocrine system has evolved to become the main control mechanism regulating spermatogenesis.
Sertoli cells express both the receptors for sex steroids and for Fsh. Therefore, Sertoli cells are the
main target of endocrine regulation of spermatogenesis. The Sertoli cells, in combination with other
somatic cells, produce the paracrine factors needed for, amongst others, maintaining the balance
between the two divisions the potential spermatogonial stem cells can go through. In other words,
these paracrine factors decide whether a stem cell goes through stem cell self renewal or through
the division into interconnected daughter cells. This balance has to be maintained in a very
conscientious manner, since tumors have to be prevented, but a lifelong stock has to be kept in order
to maintain fertility.
However, the mode of action and the identity of all the involved factors are far from complete
in vertebrates. Therefore, it is also not clear yet how hormones exactly regulate spermatogenesis via
these pathways.
In order to unravel these kinds of insecurities, the Developmental Biology group at Utrecht
University is engaged in answering two general research questions. The first problem they want to
gain insight into, is how the production and release of hormones targeting spermatogenesis is
regulated. The second problem, which is highly interlaced with the first question, is how hormones
and growth factors regulate the proliferative activity of germ cells, spermatogonial stem cells in
particular.
To approach both of these research questions, they use zebrafish (Danio rerio) as an
experimental model. This species is an established model species, since it is easy to handle, easy and
cheap to keep in large numbers, the individual developmental pathways are easy to disrupt for
experimental purposes, and the generation time is relatively fast so that developmental processes
can be studied in a short period of time. Next to that, a number of genetic tools are available since
the whole zebrafish genome has been sequenced.
Three aspects are currently brought into focus. The first is to identify candidate growth factors
that are relevant for spermatogenesis via gene expression profiling. The second is to characterize the
biological activity of the identified candidate factors by gain-of-function or loss-of-function
approaches. The last is to study the endocrine regulation of expression and release of the identified
candidate factors.
Several studies carried out by this group at Utrecht University (some of them not published
yet) demonstrated a strong effect of estrogens on zebrafish spermatogenesis. An in vivo study with
fish treated with E2, showed that E2 causes suppression of spermatogenesis. This was found by
morphological analysis of the testes of these fish. Contrasting results have been obtained by a study
conducted by de Waal et al. (2009), which state that morphological analysis of zebrafish testis ex vivo
revealed no clear disruption of spermatogenesis. These results suggest that the inhibitory effects on
spermatogenesis induced by E2 involve feedback mechanisms on the HPG-axis. Next to that, qPCR
15
analyses have been carried out by the Developmental Biology group, in order to discover whether or
not testicular gene expression was affected. It has been shown that in vivo E2 treatment caused a
modulation of gene expression of several testicular key genes. These are effects found in the fish as a
whole. Many hormonal pathways and effects operate at the same time, and are dependent on each
other for proper function. Because of these interactions, this study can not disclose whether the
gene expression in the testes is influenced directly by E2, or that E2 influences the HPG-axis, which in
turn alters testicular gene expression. In other words, it is not known yet if the observed effect of E2
is a direct or an indirect one. There is an indication that E2 has an indirect effect via the HPG-axis,
because of a former study performed by de Waal et al. (2009). This study shows that E2 has an
indirect inhibitory effect on the genes that regulate steroidogenesis in the testes. Since
steroidogenesis is closely associated with spermatogenesis, this study is indicative of an indirect
effect of E2 on the genes that regulate spermatogenesis in the testes.
However, as follows from chapter five, and as mentioned above, it is of the essence to clarify
via which pathway E2 precisely influences zebrafish testes. This can be done by studying the effects
of E2 on testes without any interference of the rest of the pathways and influences present in the
fish. In order to do so, an in vitro experiment is required.
16
Chapter 5 – Relevance
5.1 Fundamental research
This fundamental research provides insight in the way estrogens influence regulation of gene
expression in zebrafish testes. It is of the essence to know the mechanisms of action of estrogens, in
order to provide a strong theoretical fundament for studies on – for example – influences of sex
steroids on an array of tissues, effects of different substances on testicular tissue, or the molecular
pathways of gene regulation. Also, this research is a valuable addition to similar researches done in
other fish species, since the zebrafish is an established model species.
5.2 Ecotoxicology
Next to the fundamental insight that this research provides, the knowledge can be used in an
ecotoxicological framework. Several synthetically produced substances have shown to be estrogenic,
antiandrogenic or both, and are therefore able to disrupt normal endocrine function in wildlife when
released in the environment. These compounds are found in commonly used products, such as
detergents, pesticides or fungicides, paints, cosmetics, flame retardants, epoxy resins, cancer
treatment and plastics, amongst others.
Two substances, 4-tert-octylphenol and bisphenol A, are known xenoestrogens; they mimic
the action of estrogen. Octylphenol is a persistent degradation product of substances used in
pesticides, paints and cosmetics. Because of the variety of products these substances are used for,
their degradation products are commonly found in the aquatic environment. Bisphenol A is used in
plastics and epoxy resins. These epoxy resins are used, for example, to coat the inside of food cans or
as dental sealant. However, bisphenol A can leak out of this epoxy resin into food or saliva (Kinnberg
& Toft, 2003), through which it can come into the body and eventually into the aquatic environment
by the excretion of urine. Other synthetically produced estrogens may be used in contraceptives.
These estrogens are not degraded in the human body, are also emitted via urine and get into the
surface water bodies.
In this way, the concentration of estrogens in the natural environment of the fish increases
and the fish are subjected to this unnatural elevation. These high estrogen concentrations cause
male fish to lose their male characteristics and/or show a loss of fertility. It has been shown, for
example, that both abovementioned xenoestrogens caused a decrease in spermatogenetic cysts and
an increase in spermatozeugmata in enlarged sperm ducts (Kinnberg & Toft, 2003). These effects
indicate reduced or even fully blocked spermatogonial mitosis. In the study of Kinnberg and Toft, the
natural female estrogen 17-estradiol was also tested. Fish exposed to this estrogen showed an
increased number of hypertrophied Sertoli cells and efferent duct cells. All stages of spermatogenesis
were still present. However, other studies have also indicated depletion of early spermatogenetic
stages, similar to the effects of bisphenol A and octylphenol. This suggests that the effects of 17estradiol depends on the concentration (Kinnberg & Toft, 2003).
The study of Kinnberg & Toft (2003) uses concentrations of bisphenol A and octylphenol that
are not as high as those detected in the aquatic environments in which their research species, the
guppy (Poecilia reticulata) lives. It may be, however, that in other fish species the effects are elicited
at much lower concentrations than used in their study, or that the concentrations of this substances
17
is much higher in aquatic environments of other species. Furthermore, when not regulated,
concentrations of these estrogenic substances in waste water effluents may rise, through which
eventually the concentrations at which the effects reported in the study of Kinnberg and Toft are
seen, may be reached. Eventually this endocrine disruption of fish reproductive systems through
which fish lose fertility or distinctive male characteristics, may cause a decrease in the natural
biodiversity.
Evidence that this may happen, is given by comparing three populations of Common Carp
(Cyprinus carpio) living in an uncontaminated control site and in two different rivers with different
degrees of contamination with estrogenic compounds. Testes of carp living in the most contaminated
river, which had concentrations of estrogenic substances that were 3-4 times higher than in the less
contaminated river, were significantly smaller than of the carp in the less contaminated river and the
control carp. The observed concentrations in the most contaminated river were able to cause
endocrine disruption and a delay in maturational development of the testes of carp living in this
river. Furthermore, a decrease in GSI (gonadal-somatic-index; a ratio of gonad weight against body
weight) and testis weight of carp living in this river has been observed, which is also an indication of a
reduced fertility in this population. Even though this study reported that in both rivers, the
concentration of estrogenic compounds that contaminated the rivers was below the threshold to
cause observable histological changes in the testes, they still were able to elicit a disruptive effect in
fish of the more contaminated river (Hassanin et al., 2002). This may be due to a combination of
different estrogenic compounds, or estrogenic with other compounds in the environment that lower
the threshold for the estrogenic substances to have an effect. This shows that estrogenic compounds
may already affect fish in contaminated sites, even though the established laboratory threshold
concentration is not reached yet.
More research is necessary to prevent degradation products from leaking out of their original
compounds, or to replace them as a whole by non-harmful substances. Also, more research has to be
carried out in order to improve the prevention of contamination of the environment with estrogens.
In this way, the natural biodiversity of fish, and other aquatic animals, can be maintained.
5.3 Breeding
It is of importance to maintain genetic variance in a fish species to prevent inbreeding of local
populations or laboratory fish strains, through which health problems or other genetic defects may
arise. When the water bodies in which local fish populations live, get contaminated with estrogenic
compounds, a whole population might lose its fertility in a very fast manner and will neither be able
to survive as a population, nor serve as a spare gene pool for maintaining the species. When
breeding is not possible anymore, this again may affect fishery in a negative way.
Next to that, knowledge about factors influencing the HPG-axis can help solving problems in
farmed fish. There is, for example, a major problem of early puberty in several farmed fish species,
such as cod, salmonids, tilapia and sea basses. Early puberty has a negative effect on growth
performance, flesh composition, appearance, health, behavior, welfare and survival. It might also
have a genetic impact on wild populations. Also late puberty may be a problem for broodstock
management, and some species even completely fail to reach puberty altogether. Methods used
nowadays to counteract early or late puberty are not efficient or reliable enough, or have some
negative side effects (Taranger et al., 2010). The onset of puberty is controlled by the HPG-axis, so in
order to efficiently control the onset of puberty in farmed fish, it seems logical to do this via the HPG-
18
axis. For this to happen, all components involved in or influencing the HPG-axis have to be unraveled.
When all components of the HPG-axis and their molecular mechanisms, influences and effects are
known, it is possible to investigate what effects the already established methods have on these
components in order to optimize these techniques or develop new methods to improve fish
breeding.
19
Part 3: Article
Chapter 6 – Research on the influence
of estrogen on zebrafish testes
6.1 Introduction
The in vivo experiment carried out by the Developmental Biology group at Utrecht University,
as described in chapter four, does not yet clarify whether or not the changes observed are a result of
a direct effect of E2 on testicular cells, or an indirect effect mediated by the pituitary. This is because
of the many hormonal pathways and effects are working at the same time in the animal as a whole.
However, it is of great importance and relevance to know via which pathway E2 precisely influences
zebrafish testes, for example to intervene in the process of suppression of spermatogenesis.
Also the study performed by de Waal et al. (2009) does not yet clarify whether E2 has a direct
or an indirect effect on spermatogenesis. However, it gives an indication that the effect of E2 on
spermatogenesis is an indirect one, since E2 also influences steroidogenesis, a process closely
associated with spermatogenesis, in an indirect manner via the HPG-axis.
Therefore, the main goal of this research is to clarify if the effects of E2 on testicular gene
expression are direct (on testicular cells) or indirect (via the HPG-axis), in order to gain a more
detailed insight in the specific roles of estrogens on the zebrafish spermatogenesis. To achieve this
goal, an in vitro investigation is required, in order to exclude all the ways in which indirect effects
may influence gene expression, and only the direct effects will be visible. Comparison of the results
of this experiment with the results of the in vivo experiment give a final insight in whether gene
expression is regulated in a direct or indirect manner by E2.
Possible outcomes of this research are:
- There is a significant change in the gene expression when zebrafish testes are exposed in
vitro to a medium containing E2, compared to exposure to a basal medium. If this is the case,
the significant change in gene expression is then expected to be similar to the change in gene
expression seen in the in vivo E2-treated testes. This indicates that the changes seen are a
direct effect of E2, without any interference of the HPG-axis.
- There is no significant difference between the treated and basal condition in vitro. The E2treated testes and the testes exposed to a basal medium show the same levels of gene
expression. This indicates that an interaction of the hormone with the HPG-axis is necessary
to have an effect on spermatogenesis.
6.2 Material and methods
Adult male Tübingen AB strain zebrafish were used for this experiment. Animal culture,
handling and experimentation were all in accordance with Dutch national regulations, and were
approved by the Life Science Faculties Committee for Animal Care and Use in Utrecht.
A primary zebrafish testis tissue culture system as developed by Leal, de Waal, et al. (2009)
was used to study the effects of in vitro E2 treatment on testicular gene expression. After euthanizing
the fish in ice water, the two testes from each fish were excised and were incubated for 7 days. One
of both testes was kept under stimulatory conditions (receiving medium containing 10 nM E2) and
20
the other under basal conditions (receiving only tissue culture medium). In total, eleven fish were
used to perform this experiment.
After tissue culture, the testis were collected for RNA extraction and cDNA synthesis. The
changes in mRNA levels of target genes (cyp17a1, star, insl3, amh, igf1, igf3, nanos2) were
determined by qPCR assays. These genes are target genes, because they are all involved in the
process of spermatogenesis. nanos2 is found only in Aund spermatogonia and is therefore a marker of
germ cells in this developmental stage. insl3, star and cyp17a1 are found in Leydig cells, and igf1, igf3
and amh are found in Sertoli cells. star and cyp17a1 in the Leydig cells have a function in
steroidogenesis. amh, igf1 and igf3 are involved in the proliferation and differentiation of
spermatogonia. ef1 was taken as reference gene; this gene is not affected by E2 treatment and is
therefore a good indicator of the baseline of gene expression under basal and treated circumstances.
Differences between the treated and basal condition were checked for statistical significance
using the paired T-test. A significance level of P < 0,05 was applied in this statistical analysis. The
software used to perform this analysis, is the Prism4 software package (GraphPad software, San
Diego, CA, USA).
By comparing the levels of up and down regulation of genes between the in vivo and in vitro
treatments, these results give insight in the question if the effects of estrogen on zebrafish testes are
direct or indirect via the HPG-axis.
6.3 Results
As graph 6.1 depicts, the resulting Ct-values of the qPCR
analysis of the samples of ef1 lie close together. This shows
that ef1 is stable in its expression under both basal and treated
conditions, and can therefore be used as a reference gene. 18S
was also tested on stability of expression. Even though 18S was
stable (data not shown), it was not as stable as ef1, and we
therefore decided not to use 18S as a reference gene.
In graph 6.2 the gene expression of the genes of interest
under basal and E2-treated
condition is shown, as an xfold expression of the basal
gene expression given by
the reference gene ef1.
For all the genes of interest,
no significant difference
between gene expression
under basal and E2-treated
conditions was found.
Graph 6.1 – Ct-values of qPCR assays for gene ef1
under basal and E2-treated conditions
Graph 6.2 – mRNA expression of igf1, igf3, amh, insl3, star, cyp17a1 and nanos2, under basal and E2-treated
conditions, shown as an x-fold expression of the basal gene expression given by the control gene ef1
21
6.4 Discussion and conclusion
The aim of this research was to clarify if the effects of E2 on testicular gene expression are
direct, or indirect via interaction of E2 with the HPG-axis. Therefore, indirect effects via the HPG-axis
were excluded by an in vitro investigation of the testes and their gene expression under basal and E2treated conditions.
No significant difference was found between the levels of gene expression in the testes under
basal and E2-treated conditions. This indicates that E2 cannot exert its effect directly on the
testicular cells. This contrasts with the levels of gene expression observed in the in vivo study; a
significant difference was observed between basal and E2-treated conditions for the genes of
interest (unpublished results). This shows that an interaction of E2 with the HPG-axis is necessary to
change gene expression in the testis. The effect of E2 on spermatogenesis is thus an indirect one,
mediated by the HPG-axis. In this light, we might even say that the HPG-axis only consists of the HPaxis, since no direct effect of E2 on the testes has ever been found; the effects E2 has on the testes
are all mediated by the hypothalamus and/or pituitary.
Even though the results of this experiment gives a clear answer to a long-debated question, it
still does not answer in which way E2 specifically interacts with the HPG-axis. This may be via a short
loop feedback towards the pituitary, where it directly inhibits the release of Lh and/or Fsh. Another
option is for E2 to exert its influence via a long loop feedback towards the hypothalamus, where it
may inhibit Gnrh release directly, which causes a decrease in the stimulation of Lh/Fsh release, or E2
may stimulate the release of Gnih and/or dopamine, which causes a stronger inhibition of Lh/Fsh
release. This research can be used as a base for further investigations as to how E2 influences the
HPG-axis, in order to eventually answer all of the still existing questions concerning the regulation of
spermatogenesis in zebrafish.
22
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