Fish SSC

biomolecules
Review
Spermatogonial Stem Cells in Fish: Characterization,
Isolation, Enrichment, and Recent Advances of
In Vitro Culture Systems
Xuan Xie 1, * , Rafael Nóbrega 2 and Martin Pšenička 1
1 Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and
Biodiversity of Hydrocenoses, University of South Bohemia in Ceske Budejovice, Zátiší 728/II,
389 25 Vodňany, Czech Republic; [email protected]
2 Reproductive and Molecular Biology Group, Department of Morphology, Institute of Biosciences,
São Paulo State University, Botucatu, SP 18618-970, Brazil; [email protected]
* Correspondence: [email protected]; Tel.: +420-606-286-138

Received: 9 March 2020; Accepted: 14 April 2020; Published: 22 April 2020
Abstract: Spermatogenesis is a continuous and dynamic developmental process, in which a single

diploid spermatogonial stem cell (SSC) proliferates and differentiates to form a mature spermatozoon.
Herein, we summarize the accumulated knowledge of SSCs and their distribution in the testes of
teleosts. We also reviewed the primary endocrine and paracrine influence on spermatogonium
self-renewal vs. differentiation in fish. To provide insight into techniques and research related to SSCs,
we review available protocols and advances in enriching undifferentiated spermatogonia based on their
unique physiochemical and biochemical properties, such as size, density, and differential expression
of specific surface markers. We summarize in vitro germ cell culture conditions developed to maintain
proliferation and survival of spermatogonia in selected fish species. In traditional culture systems,
sera and feeder cells were considered to be essential for SSC self-renewal, in contrast to recently
developed systems with well-defined media and growth factors to induce either SSC self-renewal or
differentiation in long-term cultures. The establishment of a germ cell culture contributes to efficient
SSC propagation in rare, endangered, or commercially cultured fish species for use in biotechnological
manipulation, such as cryopreservation and transplantation. Finally, we discuss organ culture and
three-dimensional models for in vitro investigation of fish spermatogenesis.
Keywords: spermatogonial stem cell (SSC); fish; spermatogenesis; florescence-activated cell sorting
(FACS); magnetic-activated cell sorting (MACS); germ cell culture
1. Overview of Fish Germ Cell Biology

Spermatogenesis is a complex and orderly developmental process in which a single diploid
spermatogonial stem cell (SSC) proliferates and differentiates to form mature spermatozoa.
Spermatogenesis depends on the activity of the SSC, which can both self-renew to produce more
stem cells or differentiate into daughter cells committed to spermatogenesis [1–5]. The proper balance
between SSC self-renewal and differentiation is essential to assure the continuous homeostasis
of spermatogenesis. The decision as to a SSC’s self-renewal or differentiation is mediated by
cell–cell communication, and in vitro germ cell culture provides a novel platform with which to
investigate the regulatory network that determines cell fate. Furthermore, germ cell culture can be
combined with gene editing techniques such as clustered regularly interspaced shortpalindromic
repeats (CRISPR)/CRISPR-associated(Cas) for germ-line transmission, cell transplantation, nuclear
transfer, and in vitro spermatogenesis [6–9]. In the following section, we will review fish spermatogonial
cell morphology, distribution, identification, and niche, and the endocrine and paracrine regulation of
Biomolecules 2020, 10, 644; doi:10.3390/biom10040644 www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, x 2 of 31
Biomolecules 2020, 10, 644 2 of 31
and in vitro spermatogenesis [6–9]. In the following section, we will review fish spermatogonial cell
morphology, distribution, identification, and niche, and the endocrine and paracrine regulation of
spermatogenesis.
spermatogenesis. In the subsequent
In the subsequent section,
section, we will summarize
we will summarize the available protocols
the available protocols andand advances
advances
in enriching undifferentiated spermatogonia according to their unique physiochemical
in enriching undifferentiated spermatogonia according to their unique physiochemical and biochemical
and
properties.
biochemicalFinally, we will
properties. review
Finally, wedevelopments of traditional of
will review developments in traditional
vitro germ in
cellvitro
culture
germ conditions to
cell culture
maintain proliferation and survival of spermatogonia in selected fish species, as well as organ
conditions to maintain proliferation and survival of spermatogonia in selected fish species, as well as culture
and
organthree-dimensional models for in vitro
culture and three-dimensional investigation
models for in vitroofinvestigation
fish spermatogenesis.
of fish spermatogenesis.
1.1. Spermatogenesis—an Overview

1.1. Spermatogenesis—an Overview
Spermatogenesis is a continuous and dynamic developmental process which can be divided into
Spermatogenesis is a continuous and dynamic developmental process which can be divided into
three phases: the mitotic, or spermatogonial, phase with the generation of spermatogonia; the meiotic
three phases: the mitotic, or spermatogonial, phase with the generation of spermatogonia; the meiotic
phase with primary and secondary spermatocytes; and the spermiogenic phase with the haploid
phase with primary and secondary spermatocytes; and the spermiogenic phase with the haploid
spermatids emerging from meiosis and differentiating into motile, flagellated, haploid spermatozoa.
spermatids emerging from meiosis and differentiating into motile, flagellated, haploid spermatozoa.
In the spermatogonial phase, the primary increase in germ cell numbers occurs during successive
In the spermatogonial phase, the primary increase in germ cell numbers occurs during
rounds of mitotic duplication of the spermatogonia. The number of spermatogonial generations,
successive rounds of mitotic duplication of the spermatogonia. The number of spermatogonial
and hence the number of mitotic divisions before differentiation into spermatocytes, varies among,
generations, and hence the number of mitotic divisions before differentiation into spermatocytes,
but notamong,
varies within,but
species. There species.
not within, can be as few as
There cantwo (humans)
be as and(humans)
few as two as many and as 14as(guppy)
many asgenerations,
14 (guppy)
but,
generations, but, most commonly, five to eight generations are reported [10]. In invertebrates
most commonly, five to eight generations are reported [10]. In this phase, in all this phase, inandall
vertebrates, at the end of mitosis, incomplete cytokinesis occurs, and the
invertebrates and vertebrates, at the end of mitosis, incomplete cytokinesis occurs, and the two two newly-generated
spermatogonia
newly-generatedremain connectedremain
spermatogonia by a cytoplasmic
connected by bridge, instead ofbridge,
a cytoplasmic forming individual
instead cells
of forming
(Figure 1A,B). However, the cytoplasmic bridge is not present in the descendants
individual cells (Figure 1A,B). However, the cytoplasmic bridge is not present in the descendants of an SSC that
of
enters
an SSCathat
self-renewal pathway in pathway
enters a self-renewal which two in single, isolated
which two daughter
single, isolatedcells are generated.
daughter Therefore,
cells are generated.
cytoplasmic bridges are considered
Therefore, cytoplasmic bridges areaconsidered
sign of SSCadifferentiation and are present
sign of SSC differentiation during
and all subsequent
are present during
germ cell divisions (Figure 1B). All differentiated descendants of an SSC form clones
all subsequent germ cell divisions (Figure 1B). All differentiated descendants of an SSC form clones connected by
cytoplasmic
connected bybridges through
cytoplasmic which
bridges their developmental
through steps are synchronized
which their developmental (Figure 1A).(Figure
steps are synchronized These
bridges are cleaved when spermatogenesis is complete, and germ cells leave the
1A). These bridges are cleaved when spermatogenesis is complete, and germ cells leave the germinal germinal epithelium
as spermatozoa
epithelium [11].
as spermatozoa [11].
Figure 1.
Figure 1. Mitotic/spermatogonial
Mitotic/spermatogonial phase.
phase. (A) Mitosis produces
(A) Mitosis produces spermatogonium
spermatogonium clones.
clones. In
In rodents,
rodents,
type A
type A single
singlespermatogonia
spermatogonia(As) harbor
(As) harborthethe
spermatogonial
spermatogonial stemstem
cell population, whichwhich
cell population, can either
can
self-renew or generate two interconnected cells named type A-paired spermatogonia
either self-renew or generate two interconnected cells named type A-paired spermatogonia (Apr). (Apr). The A-
paired spermatogonia are interconnected by cytoplasmic bridge (CB) as a consequence of incomplete
The A-paired spermatogonia are interconnected by cytoplasmic bridge (CB) as a consequence of incomplete
cytokinesis during
cytokinesis during cell
cell division.
division. Amplifying
Amplifying divisions
divisions beyond the A-paired also do not complete
cytokinesis and continue to generate longer syncytial chains, termed
cytokinesis termed A-aligned
A-aligned spermatogonia
spermatogonia (Aal).
(Aal).
(B). An
(B). Anelectron
electron micrograph
micrograph showing
showing a cytoplasmic
a cytoplasmic bridgebridge
(arrow)(arrow) connecting
connecting daughterdaughter cells
cells resulting
resulting from spermatogonium division. The mitochondria (M), nucleus (N), smooth endoplasmic
from spermatogonium division. The mitochondria (M), nucleus (N), smooth endoplasmic reticulum (SER),
centrioles (C), and microtubules (arrowhead) are depicted in the cytoplasms of two interconnected
spermatogonia (Spg) (illustration and data: Nóbrega—unpublished observations).
During the meiotic phase, the spermatogonia differentiate into spermatocytes that go through two
meiotic divisions characterized by reshuffling of the parental genetic material during the first division
and the reduction to a haploid genome at the second division [10].
In the spermiogenic phase, the haploid spermatids emerging from meiosis differentiate into
flagellated spermatozoa without further proliferation. The morphological changes in germ cells
occurring during spermiogenesis involve reduction in cytoplasmic volume and organelles, maximum
DNA condensation, and differentiation of the flagellum, and are similar among species. However,
the final spermatozoon morphology can differ and sometimes provides taxonomic discrimination [10].
Following the general vertebrate scheme, the testes of fish are composed of the interstitial or
intertubular compartment, and the germinal or tubular compartment, separated by a basement
membrane [12]. The interstitial compartment contains the steroid-producing Leydig cells, blood/lymph
vessels, macrophages, granulocytes, and connective tissue elements [13]. The peritubular myoid
cells form a single layer of flattened cells surrounding the seminiferous tubules [14]. These cells
are contractile and involved in the transport of spermatozoa and testicular fluid in the tubule [14].
The germinal compartment is composed of germ cells at various stages of development and their
associated somatic Sertoli cells, which together form the germinal epithelium [12]. In the epithelium,
germ cell survival and development depend on constant close contact with Sertoli cells [15]. Although
many features are conserved in vertebrate spermatogenesis, the Sertoli/germ cell association differs
among vertebrates. While anamniotes (fishes and amphibians) exhibit so-called cystic spermatogenesis,
the amniotes (reptiles, birds, and mammals) present non-cystic spermatogenesis [12], in which the
seminiferous epithelium can be divided into separate stages according to the cell associations observed
in each tubular cross-section [16].
The germinal epithelium of amniote adult testes is composed of a fixed number of “immortal”
Sertoli cells that support successive waves of spermatogenesis [15,16]. During these waves, a given
Sertoli cell simultaneously supports several germ cell developmental stages (i.e., cells belonging to
different germ cell clones). The Sertoli cell base may contact spermatogonia, with lateral segments
contacting spermatocytes and early spermatids, and adluminal segments contacting late spermatids.
In this type of spermatogenesis, Sertoli cells have been shown to be terminally differentiated in the
testes, ceasing division during the early pre-pubertal phase, from approximately 10 and 20 days after
birth in mice and rats, respectively [15,17]. However, recent studies have demonstrated that Sertoli
cells from the transition region between the seminiferous tubules and the rete testis of adult testes
remain undifferentiated for a longer period and are able to proliferate, although at a lower rate [17].
In anamniote vertebrates, the germinal epithelium is composed of spermatogenic cysts. The cyst
as a morpho-functional unit is formed when a group of Sertoli cells envelope a single SSC [18].
As spermatogonia divide, the derived cells remain interconnected by cytoplasmic bridges [10,12,15].
Thus, the anaminiote Sertoli cell supports a single germ cell clone, while in amniote testes, depending
on species, at least five germ cell clones at different stages of development are supported by a single
Sertoli cell [10,12,15]. Sertoli cells from anaminiote testes are able to continuously proliferate, even
after the onset of puberty [19].
The structural differences in the Sertoli/germ cell relationship of anamniotes and amniotes result
in a less complex situation in fish than exists in mammals. Cystic spermatogenesis proceeds in a
sequential manner, while, in non-cystic spermatogenesis, several processes occur simultaneously [15].
In vertebrates, the endocrine system has evolved as the master control system of spermatogenesis,
and the somatic Sertoli, Leydig, and peritubular myoid cells became the primary targets for the major
reproductive hormones, acting as paracrine relay stations for these signals in the testes [20,21].

1.2.Biomolecules
Spermatogonial Stem Cell Niche
2020, 10, x 4 of 31
Spermatogenesisrelies
Spermatogenesis reliesononthethe activity
activity of SSCs,
of SSCs, which
which are capable
are capable of self-renewal
of self-renewal to produce
to produce more
more Spermatogenesis
stem cells or relies on
differentiation the
intoactivity
daughterof SSCs,
cells which are
dedicated capable
to of self-renewal
spermatogenesis
stem cells or differentiation into daughter cells dedicated to spermatogenesis [1–5]. The balance [1–5].to produce
The balance
between
more stem
between cells
SSC or differentiation
self-renewal and into daughter cells dedicated
differentiation the basistoof spermatogenesis the[1–5].homeostasis
The balance of
SSC self-renewal and differentiation is the basis ofismaintaining the maintaining
homeostasis of spermatogenesis.
between SSC self-renewal and differentiation is the basis of maintaining the homeostasis ofof
Ifspermatogenesis.
one process takesIfprecedence
one process takes
over the precedence overcancer
other, testicular the other, testicular
(in case cancer (in
of self-renewal) or case self-
a depletion
spermatogenesis.
renewal) or a If oneofprocess
depletion takes precedence
spermatogenesis (in over the other,
differentiation) is testicular
the outcomecancer (in case
(Figure 2). of self-
of spermatogenesis (in differentiation) is the outcome (Figure 2).
renewal) or a depletion of spermatogenesis (in differentiation) is the outcome (Figure 2).
Figure 2. Balance between spermatogonial stem cell (SSC) self-renewal and differentiation. An imbalance
Figure2.2.Balance
Figure Balance between
between spermatogonial
spermatogonial stem
stem cell
cell (SSC)
(SSC)self-renewal
self-renewaland
anddifferentiation. AnAn
differentiation.
results in testicular cancer or depletion of spermatogenesis.
imbalance results in testicular cancer or depletion of spermatogenesis.
imbalance results in testicular cancer or depletion of spermatogenesis.
The simultaneous process occurs in vertebrates showing continuous spermatogenesis, while,
Thesimultaneous
The
in seasonal simultaneous process
process
breeding species, occursfrom
occurs
a switch in vertebrates
in vertebrates showing
showing
self-renewal continuous
continuous
to differentiation spermatogenesis,
isspermatogenesis,
observed as gonadswhile, in in
while,
begin
seasonalbreeding
seasonal breedingspecies,
species,aa switch
switch from
from self-renewal
self-renewaltotodifferentiation
differentiationis is
observed
observedas as
gonads
gonads begin
begin
to mature [10].
to mature [10].
to mature [10].
Spermatogonial stem cells are maintained in a specialized microenvironment in the testes known as
Spermatogonial stem cells are maintained in a specialized microenvironment in the testes
Spermatogonial
the testicular stem3) cells
niche (Figure are maintained
[2–4,22–25]. The niche in a specialized
provides microenvironment
growth factors and cell-to-cellininteractions
the testes
known as the testicular niche (Figure 3) [2–4,22–25]. The niche provides growth factors and cell-to-
known
that as the
regulate testicular
SSC activityniche
in the(Figure 3) [2–4,22–25].
testes: cell-cycle The niche
quiescence, providesofgrowth
maintenance factors and cell-to-
the undifferentiated state,
cell interactions that regulate SSC activity in the testes: cell-cycle quiescence, maintenance of the
cell interactions
proliferation via that regulateand
self-renewal, SSCapoptosis
activity (Figure
in the testes:
3) cell-cycle quiescence, maintenance of the
[2–4].
undifferentiated state, proliferation via self-renewal, and apoptosis (Figure 3) [2–4].
undifferentiated state, proliferation via self-renewal, and apoptosis (Figure 3) [2–4].
Figure
Figure 3. Mammalian
3. Mammalian SSCSSC
niche.niche. Spermatogonial
Spermatogonial stem stem cells reside
cells (SSC) (SSC) along
residethe
along the basement
basement membrane
membrane
(BM) (BM) to
in proximity in interstitial
proximity to interstitial
Leydig cells Leydig cells
(LE) and (LE)vessels
blood and blood vessels
(BV), (BV),
and are in and are in
contact contact
with Sertoli
Figure 3. Mammalian
with Sertoli SSC
cells (SE). In thisniche. Spermatogonial
microenvironment, stem and
physical cellsparacrine
(SSC) reside alongregulate
interactions the basement
SSC
membrane (BM) in proximity to interstitial Leydig cells (LE) and blood vessels (BV), and are in contact
with Sertoli cells (SE). In this microenvironment, physical and paracrine interactions regulate SSC
cells (SE). In this microenvironment, physical and paracrine interactions regulate SSC self-renewal,
differentiation, quiescence, apoptosis, and the ability to transform into different cell types (pluripotency).
Peritubular myoid cells (PTM) are also depicted in the figure.
The niche can be defined as a microenvironment that maintains the undifferentiated state of a stem
cell by preventing its differentiation, and is usually composed of (1) supporting cells; (2) stem cells;
and (3) the surrounding extracellular matrix (Figure 3) [26–28]. In mammals, SSCs lie on the basement
membrane of the seminiferous epithelium and are in contact with Sertoli cells, which control the fate
of the SSCs via physical and paracrine interactions [4,29–31]. In addition to Sertoli cells, peritubular
myoid cells and Leydig cells may contribute soluble growth factors to the niche environment [32–34].
Mammalian SSCs are preferentially located in those areas of the seminiferous tubules near the
interstitial tissue where Leydig cells and blood vessels reside (Figure 3) [33,35–38]. Recent studies
have shown that type A undifferentiated spermatogonia are uniformly distributed on the basement
membrane of seminiferous mouse epithelium [39]. It has been demonstrated that SSC self-renewal
and proliferation are intensified in areas of high fibroblast growth factor (FGF), which corresponds to
vasculature-proximal and interstitium-proximal regions, and SSCs must be exposed to a sufficiently
high level of FGF in order to maintain the self-renewal state [40].
In anamniote species, SSCs and their niche remain poorly investigated. In fish and other
anamniotes, SSCs are single cells, not lying directly on the basement membrane, but completely
enclosed by Sertoli cell cytoplasmic extensions [10]. Similarly to mammals, fish have SSCs that are
considered type A undifferentiated spermatogonia (Aund ) [1,10]. There is evidence for two sub-types of
single, undifferentiated spermatogonia, type Aund* and type Aund , in several fish species (Figure 4A–C),
including ancient species, such as sterlet Acipenser ruthenus (Figure 4D–G) [10,22,41]. The Aund*
spermatogonia exhibit a large nucleus with little heterochromatin; a high volume of the cytoplasm; a
convoluted nuclear envelope; evident nucleoli; and particularly relevantly, darkly staining material near
invaginations of the convoluted nuclear envelope [10,41]. These patches are known as “nuage,” material
composed of ribonucleic acid (RNA) and RNA-processing proteins [10]. The Aund spermatogonia show
a smooth nuclear envelope, darker chromatin with abundant patches of heterochromatin distributed
in the nucleus, and less nuage [10,41].
These cells also exhibit differences in the cell cycle, as shown by bromodeoxyuridine
5-bromo-2’-deoxyuridine (BrdU), a marker of S-phase in pulse-chase experiments [22]. Nóbrega
and collaborators [22], using BrdU pulse-chase, reported that BrdU was rapidly diluted in type
Aund spermatogonia, while the percentage of BrdU-positive Aund* spermatogonia remained constant.
The authors [22] suggested that type Aund constitute an “active” population with rapid proliferation
and differentiation, as indicated by the more rapid loss of BrdU; Aund* are the “reserve” population
with slow self-renewal proliferation, as indicated by relatively stable BrdU labeling. Two types of
single A spermatogonia in humans display similar characteristics: a “pale” type acting as reserve,
and a “dark,” active type [42,43]. In medaka Oryzias latipes, Nakamura and collaborators [44], using a
BrdU pulse-chase experiment, also revealed distinct rapid and slow-dividing populations of oogonial
stem cells.
With respect to whether spermatogonia display a preferential distribution within the fish
testes, studies of zebrafish [22] and Astyanax altiparane [45] have shown that both Aund * and Aund
spermatogonia are located near the interstitial compartment, in contact with androgen-producing
Leydig cells and blood vessels (Figure 4A–C). These observations suggest that the androgens; growth
factors; and vascular supplies of oxygen, nutrients, and hormones may play essential roles in SSC
maintenance and self-renewal vs. differentiation in the fish testis niche [10,21].
Figure 4.
Figure Spermatogonial niche and type A undifferentiated
4. Spermatogonial undifferentiated (A und)) spermatogonia.
(Aund spermatogonia. (A) (A) Asterisks
Asterisks show
show
the most undifferentiated type A spermatogonia
the most undifferentiated type A spermatogonia (Aund* (A ), preferentially located near the interstitial
und*), preferentially located near the interstitial
compartment (delimited
compartment (delimited inin yellow)
yellow) in
in zebrafish Danio rerio.
zebrafish Danio rerio. (B,C)
(B,C) In In common
common carp Cyprinus carpio,
carp Cyprinus carpio,
both A
both Aund* andAAund
und* and und are
are locatednear
located nearthe
theinterstitium,
interstitium,close
closetotoLeydig
Leydigcells
cells(LE)
(LE)and
andblood
bloodvessels
vessels(bv).
(bv).
Sertoli cell
Sertoli cell (S)
(S) and
and type
type BB spermatogonia
spermatogonia are are shown. (D–G): Generations
shown. (D–G): Generations of of sterlet Acipenser ruthenus
sterlet Acipenser ruthenus
spermatogonia: AAund*
spermatogonia: und* and
and Aund
Aund andand
typetype A differentiated
A differentiated (Adiff(A diff ) spermatogonia.
) spermatogonia. Staining:
Staining: periodic
periodic acid–
acid–Schiff (A,E,G) and toluidine blue (B,C,D,F). Scale
Schiff (A,E,G) and toluidine blue (B,C,D,F). Scale bar: 10 µ m. bar: 10 µm.
Functional
With respectassays have spermatogonia
to whether been reported displaythat consist of transplanting
a preferential distributionSSCs fromthe
within a donor into
fish testes,
studies of zebrafish [22] and Astyanax altiparane [45] have shown that both Aund* and Aon
a recipient testis in which endogenous spermatogenesis has been blocked [46]. Depending und
self-renewal and
spermatogonia aredifferentiation
located near the capacities, transplanted
interstitial compartment, spermatogonia
in contact with are androgen-producing
able to colonize the
recipientcells
Leydig testisand
andblood
differentiate
vesselsinto functional
(Figure gametes
4A,B,C). These[46].observations
Studies havesuggest
demonstrated donor-derived
that the androgens;
spermatogenesis following type A und spermatogonia transplantation
growth factors; and vascular supplies of oxygen, nutrients, and hormones may play essential into recipient testes in roles
severalin
species
SSC of fish [22,41,47–49].
maintenance Type Avs.
and self-renewal undifferentiated
differentiationspermatogonia
in the fish testishaveniche also been shown to exhibit
[10,21].
sexual plasticity and
Functional assaysthehave
abilitybeen
to de-differentiate
reported that and differentiate
consist into oocytes
of transplanting SSCs when
fromtransplanted
a donor into intoa
zebrafish ovaries [22]. In addition, A und spermatogonia transplanted into
recipient testis in which endogenous spermatogenesis has been blocked [46]. Depending on self- sexually undifferentiated
larvae were
renewal andable to differentiate
differentiation into functional
capacities, spermatozoa
transplanted or eggs,are
spermatogonia depending on the genetic
able to colonize sex of
the recipient
the recipient [50,51]. Taken together, these findings provide evidence that
testis and differentiate into functional gametes [46]. Studies have demonstrated donor-derived SSCs represent a subset of
type Aund spermatogonia.
spermatogenesis following type Aund spermatogonia transplantation into recipient testes in several
Although
species available information
of fish [22,41,47–49]. on fish SSCs hasspermatogonia
Type A undifferentiated expanded in recent have decades,
also beenmarkers
shown tofor SSCs
exhibit
have been
sexual identified
plasticity and intheonly a few
ability to species [52], presenting
de-differentiate limitationsinto
and differentiate to detecting
oocytes whenand isolating SSCs.
transplanted
Similar to the situation in mammals, in the past decade, spermatogonium
into zebrafish ovaries [22]. In addition, Aund spermatogonia transplanted into sexually transplant was the only
means of assessing the “stemness” of putative SSCs in fish [24,48,53–55].
undifferentiated larvae were able to differentiate into functional spermatozoa or eggs, depending on
There issex
the genetic evidence
of the that promyelocytic
recipient leukemia
[50,51]. Taken zinc finger
together, theseprotein (PLZF),
findings a transcription
provide evidence thatrepressor
SSCs
essential for the maintenance of mammalian
represent a subset of type Aund spermatogonia. SSCs [56], can be a marker of SSCs in fish, as demonstrated
in the rohu Labeo
Although rohitainformation
available [57], zebrafish [58,59],
on fish SSCs dogfish
has expandedScyliorhinus
in recent canicula
decades, [60], rainbow
markers trout
for SSCs
Oncorhynchus mykiss in
have been identified [61],
onlyand several
a few speciesspecies
[52], of catfish [9,62,63].
presenting limitationsIn the neotropical
to detecting andcatfish
isolatingRhamdia
SSCs.
quelen, in situ hybridization showed that plzf is strongly expressed
Similar to the situation in mammals, in the past decade, spermatogonium transplant in type A und spermatogonia,
was the only but
was
meansalso
ofdetected
assessinginthe type Adiff spermatogonia,
“stemness” although
of putative SSCs at less
in fish intensity [63].
[24,48,53–55].
There is evidence that promyelocytic leukemia zinc finger protein (PLZF), a transcription
repressor essential for the maintenance of mammalian SSCs [56], can be a marker of SSCs in fish, as
demonstrated in the rohu Labeo rohita [57], zebrafish [58,59], dogfish Scyliorhinus canicula [60],
rainbow trout Oncorhynchus mykiss [61], and several species of catfish [9,62,63]. In the neotropical
catfish Rhamdia quelen, in situ hybridization showed that plzf is strongly expressed in type Aund
spermatogonia, but was also detected in type Adiff spermatogonia, although at less intensity [63].
Glial cell-derived
cell-derivedneurotrophic
neurotrophicfactor (GDNF)
factor is a Sertoli
(GDNF) is a cell growth
Sertoli cellfactor involved
growth factorin involved
mammalian in
SSC maintenance [64]. The GDNF-binding receptor GDNF family receptor
mammalian SSC maintenance [64]. The GDNF-binding receptor GDNF family receptor alpha1 alpha1 (GFRα1) attaches
to the membrane
(GFRα1) attaches to of the
SSCs, and is considered
membrane of SSCs, and a SSC marker in aseveral
is considered mammalian
SSC marker species
in several [64–67].
mammalian
Recently, GFRα1Recently,
species [64–67]. has beenGFRα1
detected
hasinbeen
typedetected
Aund of in Nile tilapia
type Oreochromis
Aund of Nile tilapia niloticus [68], rainbow
Oreochromis niloticus
trout
[68], [69], dogfish
rainbow [60],
trout anddogfish
[69], common[60],
carpand(Figure 5A). Incarp
common rainbow gdnfInis rainbow
trout,5A).
(Figure expressedtrout,
in germ cells
gdnf is
from spermatogonia
expressed in germ cells to spermatocytes but not
from spermatogonia to in Sertoli cells, indicating
spermatocytes that it is
but not in Sertoli not indicating
cells, secreted asthatan
autocrine SSC niche
it is not secreted factor
as an in rainbow
autocrine troutfactor
SSC niche testes,inunlike
rainbowin mammals [69].
trout testes, unlike in mammals [69].
5. (A)
Figure 5. (A) Immunoreactivity
Immunoreactivity of of GFRα1
GFRα1 waswas found
found preferentially
preferentially in
in type A undifferentiated
spermatogonia (arrowheads) of common carp. In contrast, GFRα1 immunoreactivity decreased in
differentiated
differentiatedspermatogonia
spermatogonia (arrows). The inset
(arrows). Theshows GFRα1-positive
inset undifferentiated
shows GFRα1-positive spermatogonia
undifferentiated
(arrowheads).
spermatogonia (B) POU2 was (B)
(arrowheads). detected
POU2 inwas
common carp
detected in type A undifferentiated
common spermatogonia
carp type A undifferentiated
(arrowheads),
spermatogoniawhereas its immunoreactivity
(arrowheads), whereas decreased in differentiateddecreased
its immunoreactivity spermatogonia
in (arrows). Inset
differentiated
shows POU2-positive
spermatogonia undifferentiated
(arrows). Inset shows spermatogonia.
POU2-positive = spermatocyte. Scale
Spc undifferentiated bar: 25 µm. Spc =
spermatogonia.
spermatocyte. Scale bar: 25 µ m.
Transcription factors NANOG and Pou5f3 (POU family/Oct4) are expressed in SSCs of mammals,
and Transcription
in some fish, factors
including NANOG medaka, andzebrafish,
Pou5f3 (POU Nile tilapia, and rainbow
family/Oct4) trout [61,68,70–72].
are expressed in SSCs of
Both NANOG and Pou5f3 play important roles in the maintenance and self-renewal
mammals, and in some fish, including medaka, zebrafish, Nile tilapia, and rainbow trout [61,68,70– of undifferentiated
and
72]. pluripotent
Both NANOG cellsand
[73,74]. In medaka
Pou5f3 gametes, nanog
play important roles is
inexpressed only in spermatogonia,
the maintenance and self-renewal being
of
absent in somatic cells of ovary and testis [72]. In common carp, POU2
undifferentiated and pluripotent cells [73,74]. In medaka gametes, nanog is expressed only in was detected in A und
spermatogonia,
spermatogonia,decreasing
being absent in expression
in somaticascellsspermatogonia
of ovary and differentiated
testis [72]. (Nobrega
In common et al.—unpublished
carp, POU2 was
observations)
detected in Aund (Figure 5B).
spermatogonia, decreasing in expression as spermatogonia differentiated (Nobrega
Recently, an approach
et al.—unpublished to establishing
observations) (Figuremarkers
5B). for SSC in fish has been developed [75]. The method
consisted of generating monoclonal antibodies
Recently, an approach to establishing markers for (mAb) to cell-surface
SSC in fishmolecules
has beenofdeveloped
rainbow trout
[75]. type
The
A und spermatogonia
method consisted of by inoculating
generating enriched antibodies
monoclonal live Aund spermatogonia into mice
(mAb) to cell-surface and screening
molecules with
of rainbow
atrout
combination
type Aundofspermatogonia
cell enzyme-linked immunosorbent
by inoculating enrichedassay,
livelive-cell staining, and flow
Aund spermatogonia into cytometry
mice and
(FCM)
screening[75].with
Among the obtained
a combination antibodies,
of cell two (numbers
enzyme-linked 80 and 95)assay,
immunosorbent were capable
live-cell of specifically
staining, and
labelling Aund spermatogonia
flow cytometry (FCM) [75]. Among of rainbow trout and
the obtained zebrafishtwo
antibodies, [75],(numbers
while other antibodies
80 and 95) were(numbers
capable
172 and 189) showed
of specifically strong
labelling Aundsignals for type Aofspermatogonia
spermatogonia rainbow troutand andthezebrafish
oogonia of several
[75], whilespecies
other
of salmonid(numbers
antibodies [76]. By 172
usingand these
189)antibodies
showed strong with signals
fluorescence-activated cell sortingand
for type A spermatogonia (FACS) [75] or
the oogonia
magnetic-activated
of several species ofcell sorting [76].
salmonid (MACS) [77], itthese
By using was possible
antibodiesto enrich Aund spermatogonia and
with fluorescence-activated cellincrease
sorting
transplant success rate in selected teleosts. This method presents
(FACS) [75] or magnetic-activated cell sorting (MACS) [77], it was possible to enrichthe potential to identify molecular
Aund
markers of SSCsand
spermatogonia in fish, and the
increase potentialsuccess
transplant to isolate and
rate in enrich
selectedSSCs for downstream
teleosts. This method studies, suchthe
presents as
single-cell RNA-seq or in vitro experiments. Table 1 presents the major SSC markers
potential to identify molecular markers of SSCs in fish, and the potential to isolate and enrich SSCs currently reported
in
forfish.
downstream studies, such as single-cell RNA-seq or in vitro experiments. Table 1 presents the
major SSC markers currently reported in fish.
Table 1. Spermatogonial stem cell markers in fish.

Table 1. Spermatogonial stem cell markers in fish.
Marker Specification Species Reference

SGSA-1 Spermatogonia specific-antigen-1 Japanese eel [78]
Notch1 Notch homolog protein 1 Rainbow trout [79]
Medaka [71]
Pou5/2 (Oct-4) POU domain, class 5/2
Common carp Nobrega et al. unpublished observations
Rainbow trout [80]
Ly75 (CD205) Lymphocyte antigen 75
Pacific bluefin tuna [81]
Zebrafish [58]
Carpa rohu [57]
PLZF Promyelocytic leukemia zinc finger Dogfish [60]
Rainbow trout [61]
Catfish (several species) [9,62,63]
Nile tilapia [68]
Dogfish [60]
GFRα1 GDNF-family receptor α1
Rainbow trout [69]
Common carp Nobrega et al. unpublished observations
Medaka [72]
NANOG NANOG homeobox
Nile tilapia [68]
Medaka [82]
NANOS2 NANOS homolog 2 Nile tilapia [68]
Rainbow trout [61]
NANOS3 NANOS homolog 3 Rainbow trout [61]
Rainbow trout [75]
Also applied in
Antibodies numbers 80 and 95 Not identified
Zebrafish [75]
Salmonids [77]
1.3. Endocrine and Paracrine Regulation

In vertebrates, pituitary gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone
(LH) control testicular development, and function by regulating the activity of local signaling systems
involving sex steroids and growth factors [83,84], small RNAs [85], and epigenetic switches [86].
In rodents, FSH can modulate the production of Sertoli cell growth factors that are relevant for
SSC self-renewal or differentiation [38]. Among the growth factors, the GDNF secreted by Sertoli cells
plays an important role in SSC self-renewal [64], while activin A and bone morphogenetic protein
4 (BMP4), also produced by Sertoli cells, promote differentiation [87]. Growth factors produced
by Leydig and peritubular myoid cells can also modulate SSC self-renewal or differentiation [32].
For example, colony-stimulating factor 1 secreted by Leydig and some peritubular myoid cells [32]
stimulates SSC self-renewal.
The gonadotropic hormones FSH and LH are important for testis development and
spermatogenesis in fish [10]. Despite their similar roles in vertebrates, evolution has taken a different
path for the gonadotropic hormones and their biological activities in teleost fish when compared
to the other vertebrates. Leydig cells express not only the receptor for LH, typically seen in all
vertebrates, but also the receptor for FSH [88–91]. Therefore, FSH can regulate Leydig cell functions,
including stimulation of androgen [88–90] and production of growth factors acting on spermatogonial
self-renewal and differentiation, such as Wnt5a [92] and insulin-like peptide 3 (Insl3) [93], respectively
(Figure 6). Studies have reported elevated levels of circulating androgens and FSH coinciding with
active spermatogonial proliferation in male Chinook salmon Oncorhynchus tshawytscha, and FSH
stimulates spermatogonium proliferation in juvenile Japanese eel Anguilla japonica [88] as well as
androgen production in several fish species [88–90,94]. Research in immature Japanese eels has shown
FSH-induced spermatogenesis to be blocked by trilostane, a steroid hormone synthesis inhibitor,
suggesting that FSH effects were mediated by androgens [88]. On the other hand, studies in zebrafish
have demonstrated the impact of FSH on germ cell development independent of androgens [20]. This is
supported by evidence that hundreds of testicular transcripts respond to FSH but not to sex steroids in
the testes of zebrafish [95] and rainbow trout [96]. Most of these genes belong to cellular pathways
known to regulate cell proliferation and differentiation, such as insulin-like growth factor (Igf3), Insl3,
transforming-growth factor members (Tgf-β), Wnt, Notch, and Hedgehog signaling [95]. Since Sertoli
hormone synthesis inhibitor, suggesting that FSH effects were mediated by androgens [88]. On the
other hand, studies in zebrafish have demonstrated the impact of FSH on germ cell development
independent of androgens [20]. This is supported by evidence that hundreds of testicular transcripts
respond to FSH but not to sex steroids in the testes of zebrafish [95] and rainbow trout [96]. Most of
these genes
Biomolecules 2020,belong
10, 644 to cellular pathways known to regulate cell proliferation and differentiation, such
9 of 31
as insulin-like growth factor (Igf3), Insl3, transforming-growth factor members (Tgf-β), Wnt, Notch,
and Hedgehog signaling [95]. Since Sertoli cells express FSH receptor [88–90], it is likely that Sertoli
cells express FSH receptor [88–90], it is likely that Sertoli cells act as a paracrine relay station for FSH
cells act as a paracrine relay station for FSH signal in the testes [15].
signal in the testes [15].
Figure 6. Schematic representation to summarize the regulation of zebrafish spermatogonial phase.

Figure
Fsh 6.aSchematic
exerts central role representation to summarize
in the zebrafish’s the regulation
spermatogonial phase byoftriggering
zebrafish and
spermatogonial phase.
balancing steroid
Fshgrowth
and exerts afactor
central role in theinzebrafish’s
production testicular spermatogonial phase and
somatic cells (Leydig by triggering and balancing
Sertoli cells). In Leydig steroid
cells,
and
Fsh growth factor
stimulates production
the production ofinandrogens
testicular and
somatic
Insl3,cells (Leydig
which and involved
are both Sertoli cells). Inspermatogonial
in the Leydig cells, Fsh
stimulates theand
differentiation production of androgens
proliferation. Moreover,and FshInsl3, which
promotes are both involved
spermatogonial in the spermatogonial
self-renewal by increasing
differentiation and proliferation. Moreover, Fsh promotes spermatogonial self-renewal
Wnt5a release by Leydig cells. In Sertoli cells, Fsh increases Igf3, which in turn promotes differentiation by increasing
Wnt5a
and release by activating
proliferation Leydig cells. In Sertoli
β-catenin signalingcells, Fshgerm
in the increases Igf3,also
cells. Fsh which in turn promotes
down-regulates Amh,
a differentiation
member of the TGF-βand proliferation
(transforming by growth
activating β-cateninsuperfamily
factor-beta) signaling inproduced
the germby cells. Fshcells.
Sertoli also Amh,
down-
regulates
through Amh,
PGE2 orainhibin,
memberexerts
of theanTGF-β (transforming
inhibitory growth factor-beta)
role on spermatogonial superfamily
self-renewal andproduced
germ cellby
Sertoli cells. Amh,
differentiation in thethrough
zebrafishPGE2 or To
testes. inhibin,
sustainexerts an inhibitory
its inhibitory role, role
Amhonalsospermatogonial self-renewal
decreases androgen and
andproduction
Igf3 germ cell differentiation in the zebrafish
in Leydig and Sertoli testes. To sustain its inhibitory role, Amh also decreases
cell, respectively.
androgen and Igf3 production in Leydig and Sertoli cell, respectively.
Most of the accumulated knowledge regarding the role of growth factors in spermatogonial
self-renewal
Most ofvs.
the differentiation was derived
accumulated knowledge from zebrafish
regarding the role ofstudies
growth(Figure
factors in6) spermatogonial
[20,21,89,95,97,98].
self-
The Tgf-β Amh, expressed in Sertoli cells [97–99], has been shown to exert a role in adult teleost
renewal vs. differentiation was derived from zebrafish studies (Figure 6) [20,21,89,95,97,98]. The Tgf- gonad
development in both in
β Amh, expressed males andcells
Sertoli females, particularly
[97–99], has beenat shown
germ cell
to early
exert stages
a role[100].
in adultIn zebrafish and
teleost gonad
Japanese eels, Amh
development is characterized
in both as a “spermatogenesis-preventing
males and females, particularly at germ cell earlysubstance”
stages [100]. [97,101]. Studies
In zebrafish and
ofJapanese
zebrafisheels,
have demonstrated that Amh counteracts gonadotropin-induced effects on
Amh is characterized as a “spermatogenesis-preventing substance” [97,101]. Studies of Leydig cell
steroidogenesis
zebrafish have[97], inhibits Sertoli
demonstrated thatcell
Amhpro-differentiation Igf3, and stimulates inhibitory
counteracts gonadotropin-induced effects onfactors
Leydigsuch
cell
assteroidogenesis
inhibin-α and prostaglandin E2 (Figure
[97], inhibits Sertoli cell 6) [98]. These data clearly
pro-differentiation demonstrate
Igf3, and stimulatesthat Amh inhibits
inhibitory factors
spermatogonium differentiation through modulation of growth factor production and suppression of
Leydig cell function [97,98], maintaining spermatogonia in an undifferentiated state (Figure 6).
The other well-known studied growth factor, Igf3, has been shown to be expressed exclusively in
the gonad tissue in several teleost species [21]. In the testes, Igf3 has been detected in Sertoli cells [20,102],
as well as in undifferentiated and differentiated spermatogonia, spermatocytes, and spermatids of
tilapia [102]. There is evidence that FSH stimulates Igf3, which, in turn, promotes spermatogonial
proliferation and differentiation in zebrafish testes (Figure 6) [20]. A recent study has shown that
FSH-stimulated Igf3 release activates β-cateninc signaling in type A spermatogonia to stimulate their
differentiation (Figure 6) [103].
Accumulated evidence in zebrafish demonstrates that FSH stimulates spermatogonial
differentiation by down-regulating Amh and activating β-catenin signaling via Igf3. In addition, FSH
promotes spermatogonial self-renewal through Wnt5a and the non-canonical Wnt pathways [21].
This balanced regulation could counteract depletion of Aund spermatogonia while promoting
spermatogonial differentiation (Figure 6).
2. Isolation and Enrichment of Germ Cells in Fish
2.1. Enzymatic Digestion

Isolation of germ cells by enzymatic digestion has been widely applied in both mammalian and
teleost species [104–107]. The technique involves decapsulating and mechanically mincing the gonads,
followed by dissociation via incubation with enzymes. Combinations of collagenase, trypsin-EDTA,
dispase, and DNase I are commonly used.
In fish, trypsin and collagenase are the most common enzymes employed in gonad
dissociation [108–112]. Okutsu et al. [51] reported use of 0.5% trypsin to digest testes of rainbow trout,
and the obtained spermatogonia produced viable offspring when transplanted into masu salmon
Oncorhynchus masou larvae. Lacerda et al. [48,79] dissociated testes of tilapia using collagenase, trypsin,
and DNAse I. For ovaries, enzymatic dissociation has been performed with collagenase in rainbow
trout [113] and zebrafish [114].
Variables to be considered for enzymatic dissociation include species, sex, enzyme selection and
concentration [115], and exposure time [111]. Insufficient digestion may cause low yield of single cells,
while over-digestion may lead to cell damage. Disruption of epitopes from the cell membrane surface,
which may impair germ cell function, has been observed during trypsin digestion [76,116,117]. Dispase
is reported to be a relatively mild enzyme with which to minimize damaged effects on cells [118].
In sterlet, trypsin appeared to be more effective at dissociating germ cells from gonadal tissue when
compared to collagenase [111]. More studies to address the effects of trypsin on fish germ cells
are needed.
2.2. Germ Cell Purification

Enrichment and purification are required to counteract gonad cell heterogeneity and low quantities
of spermatogonia/oogonia. Cell isolation techniques are used to separate cell populations with respect
to physiochemical and biochemical properties, including size, density, electrostatic characteristics,
and differential expressions of specific cell surface markers [119]. Germ cell purification methods have
been established to isolate homogenous populations using centrifugal elutriation [120], density gradient
centrifugation [48,121,122], differential plating [123], FACS [124,125], MACS [76], and combinations of
those methods [57,68].
2.2.1. Density Gradient Centrifugation

Discontinuous density gradients, such as Percoll and Ficoll, to separate cell populations based
on density, are commonly used for germ cell purification, and are appropriate for spermatogonia of
several fish species [10,126]. This technique has been widely applied to enrich spermatogonia before
culture or transplant in mice [48], sheep [127], primates [128], and humans [129].
In Nile-tilapia, Lacerda et al. utilized Percoll to isolate the most immature spermatogonia before
transplantation [48,79]. In loach Misgurnus anguillicaudatus [121], 60% of type A and early type
B spermatogonia were collected in the 30–36% Percoll fractions, while late type B spermatogonia,
spermatocytes, and spermatids were distributed in the 40% fraction. Wong et al. found the majority
of zebrafish spermatogonia in the 25% and 30% Percoll fractions [122]. Percoll is currently the most
commonly used method to isolate and purify fish spermatogonia [112,130,131]. However, the technique
has low resolution capacity, since it may not distinguish spermatogonia from Sertoli cells, peritubular
myoid cells, and Leydig cells. This contamination has been reported in rainbow trout [132], sterlets [111],
channel catfish Ictalurus punctatus, and blue catfish Ictalurus furcatus [62].
In contrast to what is seen during spermatogenesis, germ cells increase in size during oogenesis,
with oogonia being slightly smaller than early-stage primary oocytes [133]. The ovary also contains
a larger quantity of somatic cells. Since oogonia exhibit an intermediate size among ovarian germ
cells, their isolation is more challenging compared to that of spermatogonia. Wong et al. [114] detected
germ cell distribution in Percoll fractions in transgenic zebrafish (vasa:DsRed2-vasa). The majority
of ovarian germ cells were collected in the 25–35% Percoll interface, which provided approximately
20-fold enrichment of the initial cell suspension. The enriched cells were able to colonize the transplant
recipient gonad and produce offspring. Wong et al. [122,134] also showed that Percoll-enriched oogonia
were able to proliferate in vitro.
2.2.2. Differential Plating

Differential plating is a classic method in cell culture based on the adhesive properties of the cells.
Unlike somatic cells, SSCs will adhere to laminin or feeder cells rather than directly to a culture plate
in in vitro conditions [135]. Spermatogonial stem cells are more likely to adhere to a feeder layer after
somatic cell attachment to culture plates. Based on this attachment, differential plating can eliminate
the somatic cells. However, it is possible to lose a substantial quantity of adherent germ cells if they
are harvested at an inappropriate time. Currently, differential plating is usually combined with other
separation methods.
As an example in mammals, based on the velocity of sedimentation and differential attachment,
cell populations containing up to 70% swine SSCs were obtained, 80% of which were viable [136].
Differential plating applied in cattle can eliminate most of the somatic cells in culture [137]. The purity of
rainbow trout spermatogonia can reach >95% using differential plating [110,115]. Lacerda et al. [68,79]
combined Percoll and differential plating to enrich tilapia spermatogonia. Although differential plating
is an effective method of obtaining high purity in germ cells without causing damage, enrichment
usually takes 5–7 days, and cell properties might change during the in vitro culture period [110].
2.2.3. Flow Cytometric and Magnetically-Activated Cell-Sorting

Flow cytometry, as well as FACS, has been employed for cell sorting for more than four
decades [138]. Fluorescence-activated cell sorting is a rapid and quantitative technique with which to
examine individual cells using a range of fluorescent and light scattering signals related to cell size,
shape, granularity, surface, intracellular protein, and gene expression. When a heterogeneous group
of cells is passed through a laser system, characteristics such as morphology, viability, and surface
markers indicated by light scattering and fluorescence are captured and analyzed, and cells with
defined signals can be collected [139–141]. For some transgenic fish, target cells can be sorted by FACS
according to their self-fluorescence properties. In some endangered and cultured species in which
transgenic techniques are not suitable or available, high resolution immunoaffinity techniques are
generally conducted, based on the number and type of molecules present on the cell surface that can
be targeted by specific monoclonal antibodies conjugated with florescence or magnetic microbeads.
In mammals, FACS has been used for sorting SSCs from mouse testicular cell suspension [142].
Spermatogonial germ cells are highly enriched with characteristics such as α6-integrin positivity, c-kit
expression, low side scatter, and negative or low av-integrin expression [142]. Other SSC surface
markers that have been identified and used to sort and enrich mammalian SSCs include CD9, ITGB1,
ITGA6, GFRA1, EPCAM, NCAM1, THY1, CDH1, SSEA-4, and MCAM [143–147]. Recently, FACS has
been employed for separating spermatogonia from cancer cells in monkey testis [148], indicating a
potential application in cell therapy.
In tilapia, cell populations differentially stained with propidium iodide and carboxyfluorescein
succinimidyl ester were identified and quantified by FACS [149]. However, there are few studies of
germ stem cell surface markers. Nagasawa and colleagues [80,81,150] identified lymphocyte antigen
75 (Ly75/CD205) as a surface marker of primordial germ cells and mitotic germ cells in rainbow
trout and Pacific bluefin tuna Thunnus orientalis. However, there is still no evidence that Ly75/CD205
is capable of labeling and fractionating live type A spermatogonia. To isolate and enrich type A
spermatogonia, Yano et al. used pvasa-GFP transgenic rainbow trout and sorted cells based on diameter
and green fluorescent protein (GFP) intensity [54,151]. Further study has shown that physiochemical
characteristics such as high forward scatter and low side scatter could be used for sorting type A
spermatogonia from GFP-transgenic rainbow trout [124]. These studies showed the possibility that
spermatogonia can be dramatically enriched in a population of large cells with a simple intracellular
structure. Subsequently, Ichida et al. [125] established a method of purifying type A spermatogonia
of immature, maturing, and spermiogenic testes of Pacific bluefin tuna according to light scattering
properties using FCM. In this study, spermatogonia were enriched 15-fold compared to the unsorted
cell fraction. These findings indicate that light scattering properties are applicable to enriching
type A spermatogonia without cell-labeling systems such as transgenes and cell surface antibodies.
Monoclonal antibodies (numbers 172 and 189) have been produced by inoculating Pacific bluefin
tuna-enriched live type A spermatogonia into mice, and screened using cell-based enzyme-linked
immunosorbent assay (ELISA), immunocytochemistry, FCM, and immunohistochemistry [77]. These
antibodies were able to recognize cell surface antigens of Pacific bluefin tuna type A spermatogonia
and be used to identify live spermatogonia in a recipient following transplanattion when conjugated
with fluorescent dye [77]. This represents an important advantage in applications to commercially
valuable or endangered species that lack transgenic strains or specific molecular markers for identifying
cell lineages.
Flow cytometry is an automated, multiparametric, and sophisticated sorting tool requiring skill
and costly equipment, and it is not affordable for many laboratories. Moreover, it can require cell
labelling with fluorescent antibodies, which might alter cells and their functions.
In contrast, MACS does not require a flow cytometer and can be performed in a short period of
time through relatively simple methodology. It uses magnetic beads that bind specifically to target
cells with mAbs [152]. The dissociated testicular or ovarian cell suspension is incubated with magnetic
nanoparticles that are directly or indirectly conjugated with mAbs against a particular surface antigen.
Once bound to the intended target, beads are subjected to magnetic forces that allow immobilization of
the bound cell type and concurrent separation from other components in the suspension. Additional
washing and elution steps complete the purification cycle, resulting in an enriched preparation of a
specific cell type [152]. This technique can also be used for negative selection to eliminate undesired
cells. Compared with methods requiring FCM, MACS is a simple technique and requires no special
equipment except the magnetic beads and a magnet stand. The technique is effective for large quantities
of cells and more rapid than FCM at collecting a high number of cells [113].
Magnetically-activated cell sorting is commonly used in mammalian germ cell research [153–156],
with several surface markers employed to differentially sort spermatogonia. The c-Kit protein has
been used to separate types of spermatogonia in hamsters, mice, and monkeys [157]; GFRα1 [158],
α6-integrin [159,160], CD9 [161,162], and Thy-1 [153,163,164] have been used to isolate SSCs. Most
of these surface markers have also been applied to sorting fish spermatogonia. Panda et al. [57]
enriched highly pure type A spermatogonia from carp testis using Thy1.2 (CD90.2) antibody from
mouse. The same marker has been employed in channel catfish I. punctatus and blue catfish I. furcatus
spermatogonia [9].
It is believed that cell surface markers should exhibit low species-specificity, but mammalian
antibodies are not always suitable for work with fish spermatogonia. For this reason, Yoshizaki
and colleagues produced cell surface mAbs by inoculating enriched and live type A spermatogonia
from vasa::GFP rainbow trout into mice [75]. Using these antibodies, it was possible to isolate
undifferentiated germ cells from vasa::GFP rainbow trout with MACS [75]. The MACS-enriched cells
showed a strong GFP signal and significantly higher transplantability than the unsorted cells, especially
the ovarian cells [76]. However, like in FACS, antibodies bound to the cell surface may inhibit some
cell functions, since cell surface proteins or sugar chains could be masked by the antibodies [77].
In addition, the surface markers/antibodies developed for MACS may not be effective for all species.
It is also lower resolution than FACS, since some antibodies identified by FACS are not recognized by
MACS [147]. Therefore, more studies regarding antibody identification in fish are required.
2.3. Future Trends

Recently, microbeads and nanobeads have been used to isolate human stem cells and cancer
cells [9,165] and provide new possibilities for sorting fish germ stem cells. Microbeads and nanobeads
enable rapid binding and short labeling procedures [9,165]. Due to their small sizes, these particles do
not saturate cell epitopes and do not interfere with downstream applications. Other novel separation
methods in the stem cell field, such as aptamer-based separation [166] and affinity chromatography,
can be considered potential tools for sorting germ stem cells.
3. Germ Cell Cultures
3.1. Serum in Germ Cell Cultures

Serum is used to supply essential nutrients for in vitro cell growth [167]. Serum is a mixture
containing plasma proteins, polypeptides, growth factors, hormones, and binding proteins along with
contact and extension factors that protect cells from damage when they adhere to culture plates [168].
It may also contain unknown components that affect cell growth. The most commonly used serum
in cell culture is fetal bovine serum (FBS). Commercial media, such as Leibovitz’s L-15 medium and
Dulbecco’s modified eagle’s medium, supplemented with FBS, have become widespread in vertebrate
cell culture. In early fish germ cell culture, FBS was used to support germ cell proliferation [167]. There
is evidence that rainbow trout type A spermatogonia proliferate in culture conditions that include
a high FBS concentration in the medium, but propagation eventually ceases due to the overgrowth
of somatic cells [115]. Because rainbow trout type A spermatogonia have an extremely slow cell
cycle, somatic cells gradually occupy space and nutrition in the culture, especially at higher serum
concentrations [115]. In mice, FBS concentrations of 0.3% to 2% in media allow initial somatic cell
attachment and proliferation on flasks and subsequent SSC attachment to somatic cells with colony
formation after 5 to 7 days of culture [115]. However, when a higher concentration (5–15%) of serum
was used, SSCs propagated and formed colonies, but eventually ceased growth and detached, due to
the extensive growth of fibroblasts [169]. Thus, high serum concentrations may stimulate germ cell
proliferation, while simultaneously causing extensive growth of somatic cells, inhibiting continued
germ cell proliferation. Reports have shown that germ cells cultured with FBS were not able to
achieve long-term proliferation and maintenance of original characteristics in zebrafish testicular cell
culture [105,170]. McClusky [171] reported that FBS simultaneously stimulated both in vitro apoptosis
and [3H]thymidine incorporation in immature spermatogonia in a concentration-dependent manner.
Fetal bovine serum contains a wide range of both inhibitory and stimulatory factors [172,173]. It is
possible that germ cells and/or their associated Sertoli cells are responsive to both inhibitory and
stimulatory signals, resulting conflicting signals, and ultimately, cell death.
Bone morphogenic protein 4 (BMP-4), a stimulatory factor present in FBS [174], can induce
differentiation in cultures of zebrafish [175] and mouse spermatogonia [87,176,177]. To overcome this,
substitutes for serum are being explored. A serum-free culture of mouse SSCs was first developed
by Kutoba [163]. Mouse SSCs could maintain proliferation more than six months on minimum
essential medium-alpha supplemented with 0.2% bovine serum albumin (BSA). Recently, Aoshima
et al. demonstrated that “knockout serum replacement” medium maintained long-term growth of
SSCs in the absence of BSA and serum [178]. In fish, to suppress the overgrowth of testicular somatic
cells, Shikina and Yoshizaki replaced FBS with soluble factors, such as BSA, adenosine, and salmonid
serum [110]. The new culture extended the duration of type A spermatogonia culture, maintaining
their original morphology and GFP intensity similarly to that in vas::GFP trout spermatogonia, without
overgrowth of somatic cells [110]. In zebrafish, spermatogonia were reported to show effective
propagation for up to three months in medium with 3% FBS [179]. Wong et al. established a female
germline stem cell (FGSC) culture based on a serum-supplemented, proprietary, StemPro-34-based
(Gibco) medium, which contained the original StemPro-34 supplement plus 16 individual compounds
along with basic nutrients, including 1% FBS [122]. The proliferation of FGSCs continued for more
than 6 weeks in vitro with unchanged germ cell markers, and cells generated normal offspring after
transplantation. Multiple germ cell cultures with decreased, or no, FBS for long-term maintenance
have been reported [60,122,134]. Research should focus on development of a well-defined medium
for mixed cell populations that inhibits growth of nontarget cells while stimulating proliferation of
target cells.
3.2. Feeder Cells and Growth Factors

Fish germ stem cell survival, self-renewal, and differentiation are regulated by a combination
of intrinsic and extrinsic factors [10,97,180]. Germ cells are capable of autonomous control of the
differentiation pattern, and somatic cells, especially Sertoli cells, support germ cell development and
provide growth factors that modulate germ cell proliferation and fate. The growth factors released by
Sertoli cells are required for germ stem cell proliferation and differentiation [181], as well as initiation
of meiosis [182].
In 1994, Loir [183] cultured rainbow trout spermatogonia with L-15 supplemented with UltroserTM
serum substitute medium and 5% rainbow trout serum. In the presence of Sertoli cells, spermatogonia
survived for two weeks [183]. A co-culture system of germ cells and somatic cells was established for
the Japanese eel in 1996 [184], in which the aggregated cells undergo spermatogenesis in the presence of
11-Ketotestosterone. In 2002, using testis-derived tumor-like cells, designated ZtA6—expressing sox9a
and phagocytotic activity (Sertoli cell features)—as feeder cells, Sakai et al. reported that male zebrafish
germ cells went through mitosis and meiosis and developed into functional spermatozoa in vitro [105].
Prolongation of vasa expression in the culture suggested that ZtA6 cells promoted survival and
propagation of spermatogonia [105]. In addition, ZtA6 cells contributed to the attachment and survival
of spermatogonia at the beginning of culturing [105]. The formation of flagellated spermatozoa was
observed on day nine of culture, and clusters of spermatogonia disappeared on day 20. In this culture
system, all types of germ cells, including type A and B spermatogonia and primary and secondary
spermatocytes were cultured, precluding accurate identification of the type of germ cell stimulated
by the feeder cells. Feeder cells were heterogeneous, suggesting that somatic cells other than Sertoli
cells could be responsible for germ cell proliferation and spermatogenesis. Kurita and Sakai further
isolated and established two Sertoli cell lines (ZtA6-2 and ZtA6-12) from testis-derived tumor-like ZtA6
cells [185]. The ZtA6-2 cell line was able to support germ cell growth, and ZtA6-12 promoted germ
cell differentiation into spermatozoa [185]. The feeder cells were successfully employed in production
of spermatozoa from cultured spermatogonia [170]. The transgenic spermatozoa were differentiated
from premeiotic germ cells which were transfected with a pseudotype retrovirus in vitro. In medaka,
primary culture of testicular cells has shown formation of spermatozoa after 24 h [186]. The culture
generated both adherent and suspended cells. Proliferating cells and differentiation of spermatocytes
into spermatids or spermatozoa were observed mainly among the suspended cells. Interestingly,
the primary cells showed a dynamic equilibrium between adherent and suspended cells and could not
be separated into their respective cell populations. This study suggested that medaka germ cells are
more likely to spontaneously initiate spermatogenesis than to remain undifferentiated [186].
A normal spermatogonial cell line (SG3) derived from a mature medaka testis was established
in 2004 [108]. After 140 passages, the SG3 cell line remained stable with respect to proliferation,
karyotype, and phenotype when cultured with basic fibroblast growth factor (bFGF) and medaka
embryo extract. The SG3 cell line expressed spermatogonial stem cell genes rather than Sertoli cell
markers. In this culture system, spermatogonial cells proliferated in the absence of feeder cells,
but ceased rapid growth upon depletion of growth factors in the culture medium. The SG3 cell line
was observed to undergo meiosis and spermiogenesis to generate motile spermatozoa in vitro [108].
Similar to a previous study [186], spermatogenesis occurred in suspended aggregated germ cells.
Spermatogenesis was automatically induced by high cell confluence without subculture. It has been
suggested that bFGF may inhibit differentiation or promote self-renewal in medaka [108], indicating
that, although spermatogonia exhibit potential for continuous proliferation, their long-term culturing
depends strongly on culture conditions.
In mammalian cultures, various feeder layers have been tested. The effects of the Sandos
inbred line, mouse embryo-derived, thioguanine-resistant, ouabain resistant cells; mouse embryonic
fibroblasts; and mouse testicular stromal cells on SSC proliferation and maintenance differ between
species [87,187,188]. All these substances interfere with stability of feeder cells and make the culture
process more difficult to control. Mouse SSCs have been successfully cultivated without feeder cells on
a laminin-coated plate in the presence of GDNF [169].
As germ-cell xenotransplantation has been established for rainbow trout [55,189,190], the high
demand for germ stem cells requires an in vitro culture system to propagate and maintain spermatogonia
for long periods before transplant. Using immature rainbow trout, Shikina and Yoshizaki reported
optimized spermatogonium survival and proliferation in type A undifferentiated spermatogonia with
culture in L-15 supplemented with 10% FBS [115]. Spermatogonial proliferation was maintained
for more than one month in vitro by addition of insulin, trout embryonic extract, and bFGF to the
culture medium [115]. However, the overgrowth of testicular somatic cells restricted proliferation of
spermatogonia after one month of culture. The cultured spermatogonia colonized and proliferated
in the recipient gonads following transplantation, although at a lower rate than observed for fresh
disassociated cells [115].
To further suppress the overgrowth of testicular somatic cells and enhance the survival,
proliferation, and transplantability of spermatogonia, Shikina and Yoshizaki replaced FBS with
adenosine and salmonid serum [110]. These conditions enhanced the transplantability of cultured
spermatogonia, indicating that adenosine and salmonid serum were able to stimulate SSC survival
and self-renewal in vitro.
The development of culture media has increasingly been focused on improving germ cell expansion
while maintaining “stemness.” Initial studies expressed uncertainty as to whether germ cells in clumps
grow during in vitro culturing [105,108,110]. Several studies observed spermatogenesis after clump
formation, suggesting that differentiation may have occurred [108,186]. However, SSC proliferation
may also take the form of clumps. In L. rohita, enriched SSCs sorted by MACS with Thy 1+ were capable
of proliferation and remaining undifferentiated for more than 60 days [57]. In this feeder-free culture,
cells proliferated to form colonies with unique morphological compactness features. The induction
of proliferation was not observed in the presence of either mouse or human recombinant GDNF
in the culture medium [57]. In zebrafish, most clumps of spermatogonia were positive for BrdU,
indicating active proliferation [179]. Lacerda [68] showed that Nile tilapia spermatogonia remained
undifferentiated and able to proliferate for at least one month of culture. When purified spermatogonia
were kept at high confluence without subculture, large colonies expressing vasa, sox2, and the gfra1
were generated, without expression of meiotic marker (dmc1). Thus, it could be assumed that clumps
were likely derived from a few aggregated SSCs and expanded during proliferation [68].
Spontaneous germ cell differentiation has been observed, and probably can be attributed to
cell-cell communication [2–4]. Fish embryo stem cells and spermatogonia have been demonstrated
to differentiate in vitro via enhanced cell–cell interactions [191]. Nevertheless, it is possible that,
in contrast to in vivo, the surface properties of the cells may change in vitro and the adhesiveness
reflect specific developmental and/or cell cycle phases.
In 2012, Kawasaki et al. showed that zebrafish spermatogonia proliferate continually for longer
than one month in a culture medium supplemented with a cocktail of recombinant mammalian
growth factors, including GDNF, IGF-1, and bFGF. The cultured spermatogonia produced functional
spermatozoa after transplantation [179]. In contrast to previous studies in which GDNF showed no
effect on germ cell proliferation [57], in the zebrafish culture, Kawasaki and collaborators showed
that recombinant human GDNF enhanced spermatogonial proliferation [179]. However, compared
to mouse SSC culture, a 5 to 10-fold concentration of GDNF was needed to promote proliferation of
zebrafish spermatogonia [163,192]. In 2013, Wong et al. further studied the role of homologous growth
factors [122] in homogeneous cultures of zebrafish FGSCs. The FGSCs remained undifferentiated
for more than six weeks in vitro and showed high transplantability [122]. The FGSC proliferation
was enhanced when cultured on ovarian-somatic feeder cells (OFCs) engineered to express zebrafish
leukemia inhibitory factor (LIF), bFGF, and GDNF in a serum-free culture [122]. These conditions were
also applied to a zebrafish SSC culture [134,175] with the opposite effect observed: the number and
proliferation of spermatogonia decreased, and differentiation was stimulated after three weeks [175].
The OFCs used in this culture were assumed to express Bmp and induce spermatogonium differentiation,
since the expression of Bmp was increased in the latter part of the culture period. When blocking Bmp
signaling with the specific inhibitor dorsomorphin, spermatogonium growth could be prolonged to
6 weeks. Thus, the effect of Bmp was shown to differ in culture of zebrafish male and female germline
stem cells.
In culture of dogfish spermatogonia, GDNF has been shown to induce spermatogonium
proliferation by reducing apoptosis, and cells remained pluripotent and capable of self-renewing for
more than five months [60]. Similarly, recombinant medaka GDNFa/b enhanced the proliferative
activity of SG3 cells, which retained the spermatogonial gene expression pattern and showed alkaline
phosphatase activity [193]. There is also evidence that a high concentration of recombinant human
GDNF and LIF in culture medium was responsible for maintaining spermatogonia of sturgeon
in vitro [194]. Satoh et al. examined the in vitro effects of baculovirus-produced recombinant medaka
LIF proteins on medaka spermatogenesis [195], and found that LIF protein in the culture medium
and in co-culture with LIF-overexpressing medaka testicular somatic cells promoted spermatogonial
proliferation [195]. The influences of physiological and biochemical factors on germ cell culture have
been investigated [196,197]. Kawasaki et al. found high oxygen (20%) concentration to stimulate
spermatogenesis, while heparin contributed to propagation of spermatogonia [196].
In summary, supplementation of culture media with appropriate soluble growth factors is essential
for germ cell survival and proliferation for long-term incubation. However, in co-culture with somatic
cells, germ cells are likely to attach to somatic cells, form clumps, expand in number, and initiate
spermatogenesis. This indicates that germ cell in vitro activity may occur partly through intercellular
junctions and not only through paracrine signals from Sertoli cells. The germ cell attaches to feeder
cells prior to initiating proliferation. Some studies have demonstrated that Sertoli cell-germ cell
and germ cell-germ cell adhesive and gap junctions are involved in Sertoli cell functions to support
germ cell development within the cyst [198,199]. Thus, the role of intercellular junctions on germ cell
development, and whether there is synergism/antagonism between soluble growth factors and the cell
junctions need to be investigated.
3.3. Organ Cultures

In general, cell culture systems are not suitable for evaluating complex signal pathways among
cells and the extracellular matrix [200]. In a germ cell culture, it is difficult to control the re-association
of somatic and germ cells, and feeder cell layers may be required. In order to study gametogenesis and
its endocrine and paracrine control ex vivo, an effective organ culture system that allows germ cells to
maintain their spatial arrangement and their normal cellular and microenvironmental composition
when grown in vitro must be established. Organ culture must also maintain the integrity of the
complex regulation existing in the gonads.
The
The effort
efforttotodevelop
developa testis tissue
a testis culture
tissue system
culture beganbegan
system in the in
1960s
the in1960s
mammals [201]. Trowell
in mammals [201].
[202] developed
Trowell an organan
[202] developed culture
organ system, and Steinberger
culture system, et al. [203]
and Steinberger adapted
et al. it to rats.itIttoisrats.
[203] adapted a method
It is a
in whichintissue
method whichfragments are placed
tissue fragments on a on
are placed thin layer
a thin of agarose
layer of agarose covering
covering a aperforated
perforatedmetal metal grid
grid
(Figure 7A).
(Figure 7A).Calf
Calfserum
serum(10%)
(10%)was
was added
added to maintain
to maintain the the viability
viability ofseminiferous
of the the seminiferous
epitheliumepithelium
[203].
[203]. This agarose
This agarose gel organgel organ
culture culture
became became
a reliable a reliable
standard method standard
to studymethod to study in vitro
in vitro spermatogenesis in
spermatogenesis in mammalian species including
mammalian species including rabbits and humans [204–206]. rabbits and humans [204–206].
A B
Tissue Tissue Membrane
L-15
Agarose
gel Float
L-15
Figure 7. Organ
Figure 7. Organculture
culture systems.
systems. (A)(A)
TheThe classical
classical agarose
agarose gel model
gel model in mammals
in mammals [203]; (B)[203]; (B) the
the Japanese
Japanese
eel testis eel testismodel
culture culture model
[207]; (C)[207]; (C) the zebrafish
the zebrafish testis primary
testis primary culture[208]
culture model model[208] (1,tissue;
(1, testis testis
tissue; 2, nitrocellulose membrane; 3, agarose cylinder; 4, medium (1 mL); scale bar:
2, nitrocellulose membrane; 3, agarose cylinder; 4, medium (1 mL); scale bar: 1 cm); (D) Microfluid 1 cm); (D)
Microfluid culture model [209]. (E) Pumpless microfluid
culture model [209]. (E) Pumpless microfluid culture [209]. culture [209].
immature Japanese
Organ culturing for spermatogenesis was reported in immature Japanese eels
eels in
in the
the 1990s
1990s [207].
[207].
Similarly, to techniques use in mammals, freshly minced eel testes were placed on floats of elder pith
covered with
witha anitrocellulose membrane
nitrocellulose in tissue
membrane culture
in tissue dishesdishes
culture (Figure(Figure
7B). The basal
7B). The culture
basalmedium
culture
medium consisted of L-15 supplemented with BSA, a cocktail of amino acids, and other substances
consisted of L-15 supplemented with BSA, a cocktail of amino acids, and other substances necessary to
maintain cell activity. This culture system has been employed in analyzing effects of several steroid
hormones on eel spermatogenesis, and, with some modifications, in studies of Japanese Huchen Hucho
perryi testes and ovaries [210,211]. In 2009, a zebrafish testes culture system was developed, based on
the Japanese eel model [208]: Following disinfection, testes were placed on a 0.25 cm2 nitrocellulose
membrane in a 750 µm agarose cylinder and kept in 1 mL medium in each of 24-well flat-bottom plates
(Figure 7C). The basal culture medium was composed of L-15 supplemented with HEPES, BSA, retinoic
acid, and antibiotics [208]. The agarose cylinder was prepared using 48-well plates as molds [208].
The zebrafish testis culture model has been widely used to study endocrine and paracrine regulation
of zebrafish spermatogenesis [20,22,89,212].
A Nile tilapia testis culture model similar to the eel system was developed to study sex
differentiation in vitro [213]. A testis tissue culture system has also been established to examine
effects of FSH and LH on gene expression in rainbow trout testis [96,214]. A novel culture system
capable of effectively replicating the microcirculatory system of the mouse body was developed in
2016 [215]. The device consisted of a chamber for tissue and a channel for medium flow. The testis tissue
was separated from the flowing medium by a thin porous membrane. Nutrients and waste products
were exchanged by molecular diffusion as in vivo (Figure 7D). Using this microfluidic culture method,
mouse testis tissue could be maintained six months in vitro and could undergo spermatogenesis [215].
With the addition of appropriate hormones to the culture medium, the system can mimic in vivo
conditions. In 2018, a simpler, user-friendly system [201] using hydrostatic pressure of the medium
in a reserve tank, along with a resistance circuit, was developed to control the flow rate without the
use of a pump. The pumpless device can create culture conditions for testis tissue in a tissue chamber
(Figure 7E). These microfluidic devices have successfully created a novel organ culture system that
differs from the classic agarose gel system and has the potential to revolutionize organ culture.
The current organ culture systems described for fish can also support culture conditions suitable
for ex vivo study of testis function. Organ culture can maintain the integrity of gonadal processes
and, hence, is a powerful tool to evaluate the effects of a wide range of substances involved in fish
spermatogenesis, among them pituitary hormones, steroids, and growth factors, as well as suspected
endocrine disruptors.
3.4. Three-Dimensional Cultures

The ability to view the gonad as a three-dimensional structure, as it is in vivo, could be valuable for
study of testis development and spermatogenesis. Three-dimensional (3D) models currently represent
an optimal tool to provide a tissue-like context for cell culture. In 3D cell cultures, the communication
between cells and the scaffold is regulated by the material characteristics and scaffold properties [216].
In order to enhance cell adhesion, proliferation, and activation, materials employed in the fabrication of
scaffolds must possess characteristics involved in intrinsic biocompatibility along with the appropriate
chemistry to induce molecular recognition from cells. Three-dimensional culture systems were initially
established for clonogenic assays to explore the complex mechanisms of multipotent hematopoietic cell
proliferation and differentiation [217]. Later they were widely applied to stem cell studies, including
exploring the impact of different scaffolds on mammalian SSC culture. Lee et al. first reported the
reconstruction of the tubular structure of rat testis using collagen gel and Matrigel (BD Biosciences) [218],
obtaining germ cell differentiation with a mixture of germ cells and testicular somatic cells cultured in a
3D system. In humans, spermatocytes have been induced to differentiate into presumptive spermatids
in a collagen gel matrix culture [219]. A novel method using a soft agar culture system has become a
common method for culture of stem cells and has also been applied on mouse SSCs [220–223]. Another
3D culture model using a bicameral chamber was developed by Legendre [224]. In fish, there are
few studies of 3D culture, but fish collagen (FC) (Figure 8) is considered an ideal scaffold material
due to its biocompatibility, low immunogenicity, low cytotoxic effects, high cell viability, and high
biodegradability [225]. Studies have evaluated the FC of several species, including bester (beluga huso

huso × sterlet) and tilapia, as 3D culture scaffold materials [225–229]. To best of our knowledge, a 3D
culture
not beenofreported.
fish germThis
cellssystem
has notisbeen reported.
considered This system
a novel is considered
technique a novel
for study of technique for study
fish gametogenesis, with
of fish gametogenesis, with methods
methods and protocols to be standardized.and protocols to be standardized.
Figure 8. Scanning
Scanning electron
electron micrographs
micrographs of skin collagen, swim bladder collagen, and porcine tendon
collagen fibrils
fibrils formed
formed at 21±±1 1°C◦ Cforfor
at21 2424
h. h.
(a–c) bester
(a–c) skinskin
bester withwith
NaCl concentrations
NaCl of 0,of
concentrations 70,0,and
70,
140
and mM; (d–f)(d–f)
140 mM; bester swim
bester bladder
swim bladder with NaCl
with concentrations
NaCl concentrationsofof0,0,70,
70,and
and140
140mM;
mM; (g–i)
(g–i) porcine
collagen with NaCl concentrations of 0, 70, and 140 mM. Scale Scale bars,
bars, 55 µm
μm [227].
[227].
A three-dimensional
three-dimensional cultureculture creates
creates aa complete
complete microenvironment
microenvironment resembling
resembling the
the in
in vivo
conditions. Although
Althoughsuch such systems
systems have
have been
been shown
shown toto successfully
successfully induce spermatogenesis,
spermatogenesis,
conditions differ
conditions differfrom
from those
thosein vivo, andand
in vivo, comparisons may be
comparisons inaccurate.
may Using an
be inaccurate. appropriate
Using scaffold,
an appropriate
3D culture can avoid the ischemia that develops in long-term organ tissue culture and mimic
scaffold, 3D culture can avoid the ischemia that develops in long-term organ tissue culture and mimic the
structure
the of germ
structure cellscells
of germ within the cyst.
within the cyst.
4. Conclusions
4. Conclusions
This review
This review summarizes
summarizes the the most
most relevant
relevant aspects
aspects of of teleost germ cell
teleost germ cell biology,
biology, including
including
spermatogonium characterization, and novel techniques for isolation and
spermatogonium characterization, and novel techniques for isolation and culture. Combined culture. Combined with with
such
procedures as cryopreservation and transplantation, stem germ cell propagation
such procedures as cryopreservation and transplantation, stem germ cell propagation can be a can be a powerful
tool for research,
powerful tool for aquaculture, and conservation
research, aquaculture, of endangered
and conservation species (Figure
of endangered 9).(Figure
species Stem germ cell
9). Stem
isolation,
germ cellenrichment, and culture conditions
isolation, enrichment, and culture to support theirto
conditions proliferation
support theirwithout differentiation
proliferation will
without
help to overcome the limitations of low germ cell numbers for cryopreservation
differentiation will help to overcome the limitations of low germ cell numbers for cryopreservation and transplantation
(Figure 9). Germ cell culturing
and transplantation (Figure is9).valuable
Germ forcellstudying
culturingthe is
mechanisms
valuable for underlying
studyingmitotic
the proliferation,
mechanisms
differentiation of stem cells, meiosis, and stem cell regulation in vitro. Of particular
underlying mitotic proliferation, differentiation of stem cells, meiosis, and stem cell regulation interest is the
in
potential to combine germ cell culture with gene editing techniques such as CRISPR/Cas
vitro. Of particular interest is the potential to combine germ cell culture with gene editing techniques for germ-line
transmission
such by cell transplantation,
as CRISPR/Cas nuclear transfer,
for germ-line transmission by celland/or in vitro spermatozoon
transplantation, production
nuclear transfer, and/orforin
artificial insemination. Knowledge in this area has evolved rapidly, and
vitro spermatozoon production for artificial insemination. Knowledge in this area has evolved has revealed novel and
promising
rapidly, and approaches
has revealed to germ
novelstem
and cell manipulation.
promising approaches to germ stem cell manipulation.
Dissociation of FACS Percoll MACS

gonadal cells
Enrichment of germ cells
Characterization of
germ cell populations
Purified germ stem cells
liquid
nitrogen
Transplantation
In vitro
spermatogenesis
Gene editing
In vitro expansion of
germ stem cells
Figure 9. Potential
Figure applications
9. Potential of germ
applications cell isolation
of germ and culture.
cell isolation Testes are
and culture. removed
Testes from the from
are removed fish body
the fish
and body
dissociated, providing a cell suspension that can be sorted using fluorescence-activated cell sorting
and dissociated, providing a cell suspension that can be sorted using fluorescence-activated cell
or centrifugation in a density gradient; e.g., in Percoll solution or by magnetic-activated cell sorting.
sorting or centrifugation in a density gradient; e.g., in Percoll solution or by magnetic-activated cell
The purified germ cell suspension can be characterized with respect to molecular characteristics and
sorting. The purified germ cell suspension can be characterized with respect to molecular
morphology. Purified cells may be injected into a recipient to generate germ-line chimera or amplified
characteristics and morphology. Purified cells may be injected into a recipient to generate germ-line
by in vitro culture. Cultured cells can be employed in gene editing or subjected to cryopreservation.
chimera or amplified by in vitro culture. Cultured cells can be employed in gene editing or subjected
to cryopreservation.
Author Contributions: Conceptualization, M.P.; methodology, X.X. and R.N.; writing—original draft preparation,
X.X.Author
and R.N.;Contributions:
writing—review Conceptualization,
and editing, M.P. andM.P.; methodology,
R.N.; supervision, X.X. andR.N.;
M.P. and R.N.;project
writing—original
administration,draft
M.P.preparation,
and R.N.; funding acquisition,
X.X. and M.P. and R.N. and
R.N.; writing—review All authors
editing,have
M.P.read
andand agreed
R.N.; to the published
supervision, M.P. andversion of
R.N.; project
the manuscript.
administration, M.P. and R.N.; funding acquisition, M.P. and R.N. All authors have read and agreed to the
M.P. version
published
Funding: and X.X.ofwere financially supported by the Ministry of Education, Youth and Sports of the Czech
the manuscript.
Republic—project CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370); and the Czech
Funding:
Science M.P. and
Foundation X.X.
(grant were financially
number 20-23836S).supported
R.N. was by the Ministry
supported by Sãoof Education, Youth
Paulo Research and Sports(FAPESP)
Foundation of the Czech
Republic—project CENAKVA (LM2018099) and Biodiversity (CZ.02.1.01/0.0/0.0/16_025/0007370); and the Czech
(14/07620-7).
Science Foundation
Acknowledgments: The(grant number
authors would20-23836S). R.N. Beatriz
like to thank was supported
Marques byde
SãoSouza
Paulofor
Research Foundation
the technical (FAPESP)
support in
(14/07620-7).analysis of sturgeon gonads, and Dr. Angel Andres Arias Vigoya for the GFRα1 and POU2
the histological
immunodetection in common carp testes. We would like to thank Alan Pike and Kathleen Hills for English
Acknowledgments:
correction. We are alsoThe authors
grateful to would like toreviewers
anonymous thank Ms. forBeatriz
theirMarques
valuablederecommendations
Souza for the technical support in
to improve
the histological
the manuscript. analysis of sturgeon gonads, and Dr. Angel Andres Arias Vigoya for the GFRα1 and POU2
immunodetection in common carp testes. We would like to thank Mr. Alan Pike and Ms. Kathleen Hills for
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
English
study; in thecorrection.
collection,We are alsoorgrateful
analyses, to anonymous
interpretation of data; reviewers for their
in the writing valuable
of the recommendations
manuscript; to improve
or in the decision to
the manuscript.
publish results.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
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biomolecules

Abstract: Spermatogenesis is a continuous and dynamic developmental process, in which a single

1. Overview of Fish Germ Cell Biology

Biomolecules 2020, 10, 644; doi:10.3390/biom10040644 www.mdpi.com/journal/biomolecules

1.1. Spermatogenesis—an Overview

Biomolecules 2020, 10, x 4 of 31

Table 1. Spermatogonial stem cell markers in fish.

Table 1. Spermatogonial stem cell markers in fish.

Marker Specification Species Reference

1.3. Endocrine and Paracrine Regulation

Figure 6. Schematic representation to summarize the regulation of zebrafish spermatogonial phase.

2. Isolation and Enrichment of Germ Cells in Fish

2.1. Enzymatic Digestion

2.2. Germ Cell Purification

2.2.1. Density Gradient Centrifugation

2.2.2. Differential Plating

2.2.3. Flow Cytometric and Magnetically-Activated Cell-Sorting

2.3. Future Trends

3. Germ Cell Cultures

3.1. Serum in Germ Cell Cultures

3.2. Feeder Cells and Growth Factors

3.3. Organ Cultures

3.4. Three-Dimensional Cultures

Biomolecules 2020, 10, x 19 of 31

Dissociation of FACS Percoll MACS

Purified germ stem cells

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