The Role of Retinoic Acid (RA) in Spermatogonial
Differentiation 1
Authors: Busada, Jonathan T., and Geyer, Christopher B.
Source: Biology of Reproduction, 94(1)
Published By: Society for the Study of Reproduction
URL: https://doi.org/10.1095/biolreprod.115.135145
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BIOLOGY OF REPRODUCTION (2016) 94(1):10, 1–10
Published online before print 11 November 2015.
DOI 10.1095/biolreprod.115.135145
Minireview
The Role of Retinoic Acid (RA) in Spermatogonial Differentiation1
Jonathan T. Busada3 and Christopher B. Geyer2,3,4
3Department of Anatomy and Cell Biology, Brody School of Medicine, East Carolina University, Greenville,
North Carolina
4East Carolina Diabetes and Obesity Institute, Brody School of Medicine, East Carolina University, Greenville,
North Carolina
ABSTRACT The production of mammalian spermatozoa in the testis is a
stem cell-based developmental process. Each adult mouse testis
Retinoic acid (RA) directs the sequential, but distinct, contains approximately 3000 unipotent spermatogonial stem
programs of spermatogonial differentiation and meiotic differ- cells (SSCs) that either self-renew or initiate spermatogenesis
entiation that are both essential for the generation of functional by producing undifferentiated progenitor spermatogonia that
spermatozoa. These processes are functionally and temporally
are destined to enter meiosis [4, 5]. This small population of
decoupled, as they occur in distinct cell types that arise over a 9
week apart, both in the neonatal and adult testis. However, our SSCs is responsible for the production of 10 sperm per day
understanding is limited in terms of what cellular and molecular throughout the male mouse reproductive lifespan [6]. The
changes occur downstream of RA exposure that prepare decision to remain a stem cell or to proliferate and differentiate
differentiating spermatogonia for meiotic initiation. In this is crucial for the reproductive health of the male. In humans,
review, we describe the process of spermatogonial differentia- insufficient or excessive differentiation can result in reduced or
tion and summarize the current state of knowledge regarding RA lost sperm production (nonobstructive oligo- or azoospermia),
signaling in spermatogonia. which are leading causes of male infertility. Strikingly, there
appears to be a decrease in overall male reproductive fitness in
developmental biology, differentiation, gonocyte, prosperma-
Western societies over the past several decades, which has been
togonia, retinoic acid, retinoids, spermatogenesis, spermatogonia,
termed testicular dysgenesis syndrome. This syndrome is
testis
thought to result from environmental changes and is charac-
terized by a decline in semen quality, increases in hypospadias
INTRODUCTION
and cryptorchidism, and an increase in the incidence of
Multicellular organisms contain a wide variety of special- testicular cancer [7–9]. Undifferentiated male germ cells
ized cell types that originate from less specialized cells by (specifically, primordial germ cells, prospermatogonia, and
cellular differentiation, which involves a progression of potentially, spermatogonia) that fail to properly differentiate
specific changes that prepare them for their ultimate function. are hypothesized to be the basis for carcinoma in situ, the
Many specialized cells have a finite lifespan and therefore must precursor to most forms of testicular cancer [10–12].
be periodically replaced by a population of uni- or multipotent Significant effort has been exerted to understand how the
adult stem cells. These stem cells balance self-renewal with the foundational SSC population is maintained, and a number of
production of progenitor cells that proliferate to amplify their excellent recent reviews document this progress [13–18]. In
numbers before committing to a specific cell fate. As an contrast, little is known about the cellular changes accompa-
example, the consistent daily production of 1012 blood cells in nying spermatogonial differentiation, and the pathways and
the adult mammalian bone marrow is accomplished by a proteins involved remain poorly defined. The purpose of this
comparatively small population of hematopoietic stem cells review is to provide a developmental perspective on the current
(estimates range from approximately 16 800 to 81 000), the state of knowledge about the essential program of spermato-
progenitors of which follow unique programs of differentiation gonial differentiation that prepares undifferentiated progenitor
to become leukocytes, erythrocytes, or megakaryocytes [1–3]. spermatogonia for entry into meiosis.
1Supported by a grant from the NIH/NICHD (HD072552 to C.B.G.). SPERMATOGONIAL BIOLOGY
2Correspondence: Christopher B. Geyer, Brody School of Medicine at
East Carolina University, 600 Moye Blvd., Greenville, NC 27834. Following sex determination in the fetal mouse testis,
E-mail: geyerc@ecu.edu prospermatogonia (also termed gonocytes) proliferate until
approximately Embryonic Day (E) 14.5 and then enter a
Received: 1 September 2015. mitotically quiescent state in G of the cell cycle until after0
First decision: 29 September 2015. birth [19, 20]. Prospermatogonia then re-enter the cell cycle at
Accepted: 6 November 2015. approximately Postnatal Day (P) 1–2 and migrate from the
 2016 by the Society for the Study of Reproduction, Inc. This article is center to the periphery of the testis cords; completion of both
available under a Creative Commons License 4.0 (Attribution-Non- tasks is apparently required for their survival [21]. Prosper-
Commercial), as described at http://creativecommons.org/licenses/by/
4.0/. matogonia become spermatogonia at approximately P3–P4, as
eISSN: 1529-7268 http://www.biolreprod.org they migrate to the periphery of the testis cords and become
ISSN: 0006-3363 flanked by somatic Sertoli cells within the testis cord and
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BUSADA AND GEYER
FIG. 1. Germ cell differentiation in the neonatal testis. A) At P1, prospermatogonia are located in the center of the testis cords with adjacent Sertoli cells
and peritubular myoid (PM) cells surrounding the tubules. B and C) By P4, spermatogonia have become either A (GFRA1ώ, in green) or A (STRA8ώ,
undiff diff
in red). In C, the testis cords are outlined by F-actin staining with phalloidin (in blue). Bar Ό 10 lm.
peritubular myoid cells that surround the outside of the cord are termed A (A ), whereas those connected by an
single s
(Fig. 1, A and B). The proteins and signaling networks intercellular bridge are termed A (A ). As progenitor
paired pr
involved in this transition are currently under investigation by spermatogonia undergo transit-amplifying divisions, they form
several laboratories. It has recently been shown that suppress- progressively longer interconnected chains as A (A )
aligned al
ing NOTCH signaling in Sertoli cells is important for spermatogonia. Although A , A , and A spermatogonia are
s pr al
maintaining quiescence in prospermatogonia [22–24]. Also, all classified as undifferentiated, evidence supports a diminu-
two reports indicate the requirement for the chromatin- tion of stem cell capacity that accompanies increased chain
modifying protein Swi-independent 3a (SIN3A) in regulation length (for review, see [17]). However, evidence indicates that
of mitotic re-entry [25, 26]. Members of the transforming some A spermatogonia, termed false pairs, represent SSCs
pr
growth factor beta (TGF-b) superfamily, such as the activins, that have divided but not moved away from one another (for
inhibins, and bone morphogenetic proteins (BMPs), have likely review, see [13]).
roles in the initiation of spermatogenesis, although their The commitment to enter meiosis is made with the transition
requirement in vivo requires further study (for reviewed, see of A into A spermatogonia. This transition requires
undiff diff
[27]). retinoic acid (RA), which will be discussed in detail below. The
This initial neonatal spermatogonial population is hetero- first differentiating spermatogonia are termed type A , which
1
geneous, and both undifferentiated (A ) and differentiating undergo five subsequent divisions to form A , A , A ,
undiff 2 3 4
(A ) spermatogonia are detectable as early as P3–P4 (Fig. intermediate (In), and finally, type B spermatogonia before
diff
1C) [28–31]. The origin of this heterogeneity is currently becoming preleptotene spermatocytes that are in the first phase
undefined, although it is apparent that spermatogonia are of meiosis I. During differentiation, the cell-cycle duration
capable of differentially responding to extrinsic signals from decreases, and a significant amount of germ cell loss occurs,
somatic cells, which will be discussed in greater detail below. such that only an estimated 39% of the expected numbers of
A small percentage of the A population contains the future preleptotene spermatocytes are formed [36–38]. It is important
undiff
SSC pool, which functions to support steady-state spermato- to note that male germ cells must complete this prolonged
genesis throughout the remainder of the male reproductive stepwise differentiation process that takes approximately 1 wk
lifespan. The rest of the surviving neonatal spermatogonia in order to gain competence to enter and successfully complete
become progenitor or differentiating spermatogonia that will meiosis. In the fetal testis, prospermatogonia respond to
enter meiosis beginning at approximately P10 to give rise to precocious RA exposure by inappropriately expressing meiotic
the first fertilizable sperm that are seen around P35. It is markers before rapidly dying by apoptosis [39–41]. In the
presumed that the first differentiating spermatogonia arise neonatal and adult testis, A spermatogonia exposed to
undiff
directly from prospermatogonia without first forming an SSC exogenous RA cannot be hastened to enter meiosis without
[29, 30, 32]. This transition (prospermatogonia to type A progressing through these steps [42–46].
spermatogonia) marks the ‘‘initiation of spermatogenesis’’ in The accurate identification of spermatogonial subtypes at
the mouse. the histological level takes a considerable amount of
During steady-state spermatogenesis, the products of SSC experience, and it relies on characteristic differences in nuclear
divisions either maintain the stem cell pool (self-renewal shape and diameter as well as heterochromatin appearance in
division) or generate progenitor spermatogonia that will paraffin-embedded testis sections carefully prepared with
proliferate and differentiate to eventually enter meiosis. certain fixatives (e.g., 5% glutaraldehyde or Bouin solution).
Daughter cells of an SSC division that are destined to In the adult testis, identification is aided by the fact that germ
differentiate retain a relatively large (;1 lm diameter) tubular cells are present in defined stages of the seminiferous
connection, termed an intercellular or cytoplasmic bridge, that epithelium [29, 47]. Neonatal and juvenile testes lack clearly
results from incomplete cytokinesis [33, 34]. The function of defined epithelial stages, although some have proposed that
these bridges is unclear. However, they are highly conserved staging is possible beginning with the appearance of prelepto-
through evolution and allow sharing of molecules and even tene spermatocytes at approximately P8. The absence of
organelles such as mitochondria between cells within a defined stages makes it difficult to impossible to discriminate
syncytium (for review, see [35]). This likely aids in the reliably between all spermatogonial subtypes at the light
synchronization of subsequent divisions, and it may also microscopic level based on morphology alone. This is because
provide for the sharing of essential X-linked gene products differences in morphology (especially of chromatin) are both
between adjacent X- and Y-bearing haploid postmeiotic subtle and quite variable [28, 48]. The topological arrangement
spermatids later during spermiogenesis. Single spermatogonia of A , A , and A spermatogonia can be visualized in whole
s pr al
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SPERMATOGONIAL DIFFERENTIATION IN THE MOUSE
Three types of spermatogonia are present in the human
testis—namely, types A , A , and B. Prospermatogonia
dark pale
transition into both A and A spermatogonia by 2–3 mo
dark pale
after birth. The first differentiating type B spermatogonia are
visible by 4–5 yr of age but only represent approximately 10%
of the spermatogonial population by age 10 [61]. It is currently
held that A spermatogonia are non- or slow-cycling and
dark
represent the ‘‘reserve’’ stem cell population that can be
activated following damage to the germ cell population. In
contrast, Apale spermatogonia are analogous to the Aundiff
population in rodents and can be present as single cells or
longer chains of interconnected cells (for review, see [62]).
EXTRINSIC SIGNALS DIRECT SPERMATOGONIAL FATE
Tissue homeostasis is maintained in many epithelial tissues
that have a high rate of turnover through a delicate balance
between stem cell self-renewal and the production of
progenitors that proliferate and differentiate. In the testis,
distinct signals received by developing male germ cells direct
this balance. Numerous studies have revealed that the
FIG. 2. Selected spermatogonial protein fate markers. The estimated undifferentiated state is maintained by the binding of the
detection level for each marker is listed by a plus sign (ώ, ώώ, or ώώώ). ligands provided by Sertoli and/or peritubular myoid cells to
Levels are subjective, and comparisons are only valid for tissues prepared, their cognate receptors on spermatogonia both in vivo and in
incubated, and stained similarly. The minus sign () indicates that the vitro. While numerous additional signaling pathways surely
listed protein is undetectable but not necessarily absent from the listed cell
type. *Inferred (largely studies using fluorescent reporter constructs in await discovery, the best described ligand/receptor pairs
transgenic mice). currently include glial cell-derived neurotrophic factor (GDNF)
to GFRA1/RET [63–70], chemokine (C-X-C motif) ligand 12
(CXCL12) to CXCR4 [71], and fibroblast growth factors 2 and
mounts of seminiferous cords or tubules, but the germ cells 8 (FGF2 and FGF8, respectively) to FGFR [72–77].
must be labeled either by transgenic expression of a fluorescent Spermatogonial differentiation requires all-trans retinoic
reporter such as GFP or by using indirect immunofluorescent acid (ATRA; referred to as RA), the bioactive oxidative
antibody staining, as described below. metabolite of vitamin A/retinol [46, 78–81]. Spermatogonia
Another useful tool for spermatogonial identification is cannot progress past the A stage in mice when RA signaling
al
labeling with antibodies against specific protein markers that is blocked following prolonged consumption of a vitamin A-
have been linked to cell fate or function (Fig. 2). A number of deficient (VAD) diet or administration of specific compounds
characteristic proteins have been identified that are detectable (e.g., bis-[dichloroacetyl]-diamines such as WIN 18 446) that
primarily in A spermatogonia in the adult testis, including restrict the synthesis of RA from retinal by inhibiting
undiff
ID4, GFRA1, RET, ZBTB16/PLZF, CDH1 and PAX7 [17] as retinaldehyde dehydrogenases [82–84]. Either treatment causes
well as NANOS2 and NANOS3 (which are indirectly labeled arrested spermatogonial differentiation at the A -to-A transi-al 1
as FLAG-tagged transgenes) [49, 50]. In contrast, KIT and tion that can be reversed by retinoid supplementation, resulting
STRA8 are the only two markers currently detectable in in resumption of spermatogenesis and restoration of fertility
differentiating, but not undifferentiated, spermatogonia [51– [79, 82, 83, 85]. Although the primary role for RA in the testis
53]. Although STRA8 is generally considered to be a nuclear in directing spermatogonial differentiation is clearly estab-
protein, it has also been detected in the cytoplasm [54, 55]. Its lished, the mechanisms activated downstream of RA exposure
function is currently unknown, but its determination should are largely undefined.
help clarify this observation. For A subtypes (A , A , A , or
diff 1 2 3
A ), no protein markers are currently identified that allow them REGULATING SPERMATOGONIAL EXPOSURE TO RA
4
to be distinguished from one another without knowing the Cellular exposure to RA is managed at multiple levels by
stage of the adult seminiferous tubule within which they reside. proteins that regulate its synthesis, reception, storage/transport,
Some protein markers have increased levels in differentiating and degradation [85–87]. Several laboratories are currently
spermatogonia, including RHOX13, SOHLH1, and SOHLH2 focused on understanding how RA is distributed within the
[42, 56–60]. Whereas the above markers are restricted to either testis such that only A spermatogonia respond to this
undifferentiated or differentiating spermatogonia in the juve- diffdifferentiating signal. Two general scenarios can be envisioned.
nile and adult testis, this is largely not true in the neonatal In option 1, all spermatogonia are primed to respond to RA, but
testis. We recently reported significant overlap, in that markers the exposure to RA is tightly controlled; in option 2, all
for A colocalized with the spermatogonial differentiation
undiff spermatogonia are exposed to RA, but only some can respond.
marker KIT (as well as STRA8) through approximately P10, Current evidence in the literature suggests that both scenarios
with GFRA1 and RET being notable exceptions [44]. This are involved (see Fig. 3A). In support of this notion, the
suggests that the differentiating program is overlaid on the postnatal deletion of single, seemingly key molecules involved
undifferentiated state in prospermatogonia and spermatogonia in RA reception, storage, and degradation in knockout (KO)
in the neonatal testis. Whereas these fates appear more clearly mouse models has not thus far resulted in phenotypes that fully
established in the juvenile and adult, transient overlap of some recapitulate the VAD model’s arrested spermatogonial differ-
undifferentiated and differentiating markers also occurs in adult entiation and infertility [88–96]. This indicates that both
spermatogonia. exposure and reception are parts of a complementary system
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BUSADA AND GEYER
FIG. 3. Regulating spermatogonial exposure to RA. A) Two options, described in the text, for how spermatogonia become exposed to or avoid RA. In
option 1, an SSC contains RARs (yellow Y) and so could presumably differentiate in response to RA (blue triangle). However, expressed CYP26 (orange T)
catabolizes RA and prevents binding to RARs. In option 2, an SSC that does not express RARs is pictured. Therefore, although the cell has access to
abundant RA, it does not differentiate because it lacks the requisite RARs to transduce the signal. B) Two type A spermatogonia within the same cord adopt
different fates based on their response to RA. The upper left spermatogonium remains GFRA1ώ (green) and can respond to GDNF but does not respond to
RA. This may result from lack of requisite RARs and/or degradation by CYP26 enzymes active in either the spermatogonium itself or in the RA-producing
cell (adjacent Sertoli). The lower right spermatogonium responds to RA by becoming STRA8ώ (red) and KITώ (not shown) and differentiating. This may
result from gain of RAR expression or the absence of localized CYP26-mediated degradation within the spermatogonium itself or adjacent Sertoli cells.
with redundant controls built in to ensure spermatogonia of spermatogenesis in young mice (age, ,7 wk); after that, KO
respond appropriately to RA. males exhibit normal fertility and testis histology. These results
Results from several reports support a role for regulated RA clearly suggest that the first step in the synthesis of RA (retinol
exposure (option 1 above) in maintaining spermatogonial cell to retinal) is performed by another retinol dehydrogenase
fate. In the fetal testis, quiescent prospermatogonia must be during steady-state spermatogenesis in the adult. In addition,
protected from RA exposure or they will begin to differentiate three retinaldehyde dehydrogenases (Aldh1a1–3, previously
and enter meiosis precociously and, as a result, die by termed Raldh1–3) have been conditionally deleted in mouse
apoptosis [40, 41]. This protection is provided, at least in part, Sertoli cells [45]. Spermatogonia in these mice fail to
by the RA-degrading action of the cytochrome P450 enzyme differentiate; however, injection of RA or a retinoic acid
CYP26B1 [39, 94]. After birth, a subset of spermatogonia receptor (RAR) A-selective agonist reinitiates spermatogenesis.
becomes exposed to RA by P3–P4 (as evidenced by their There is also evidence supporting a role for regulated RA
expression of the RA-inducible Stra8 gene) [42, 53, 58, 84, reception (option 2 above) in maintaining spermatogonial cell
97]. If CYP26B1-mediated degradation is responsible for fate. RA is a lipid-soluble molecule that enters the cell to bind
protecting a subset of postnatal spermatogonia from RA with high affinity to its cognate receptors. The RAR has three
exposure, this implies that degradation activity is reduced or isotypes (RARA, RARB, and RARG), and each is capable of
lost near STRA8ώ A spermatogonia, although this has not heterodimerizing with a retinoid X receptor isotype (RXRA,
diff
been shown experimentally. In the adult testis, the majority of RXRB, or RXRG). The RAR isotypes have distinct expression
A spermatogonia transition to differentiating KITώ A patterns in the testis: RARB is undetectable, RARA predom-
undiff 1
spermatogonia at stage VIII of the seminiferous epithelial inates in Sertoli cells, and RARG predominates in Adiff
cycle. This coincides with STRA8 induction in A spermato- spermatogonia in the neonatal, juvenile, and adult testis [92,
diff
gonia and preleptotene spermatocytes [54, 98], and it was 102]. Therefore, based on this expression pattern, it is logical to
recently shown that a pulse of RA peaks at stage VIII [51]. assume that RA signaling through RARG directs spermatogo-
Therefore, RA levels are clearly modulated during steady-state nial differentiation. Whole-body as well as germ cell and
spermatogenesis in the adult; epithelial stages VII–VIII, which Sertoli cell KO mice have been generated with deletions of
are exposed to the highest levels of RA, contain germ cells each of the RAR isotypes to address their respective roles in the
undergoing the three processes that are dependent upon RA testis. Rarb-null mice are viable and fertile, with no apparent
(spermatogonial differentiation, meiotic initiation, and spermi- defects in spermatogenesis [103], which is expected based on
ation) [46, 79, 99, 100]. its apparent lack of expression in the testis. In contrast, both
Evidence supports a requirement for the production of RA Rara-null and Rarg-null mice exhibit varying defects in
by Sertoli cells. Circulating retinol is converted into RA by two spermatogenesis, although loss of either gene singly or in
successive reactions: retinol to retinal by retinol dehydroge- combination does not recapitulate the VAD phenotype of
nases, and retinal to RA by retinaldehyde dehydrogenases. The blocked spermatogonial differentiation [88–90, 92, 104, 105].
conditional deletion of retinol dehydrogenase 10 (Rdh10) in Although Rara KO mice are infertile [88, 89, 105], deletion of
Sertoli plus germ cells, and, to a lesser extent, in Sertoli cells Rarg has no obvious effect on spermatogonial differentiation
only, resulted in loss of A differentiating spermatogonia [101]. during the first wave of spermatogenesis, and many tubules are
1
Interestingly, this defect is only manifest during the first wave apparently normal until KO mice reach advanced age (;12
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SPERMATOGONIAL DIFFERENTIATION IN THE MOUSE
mo) [92]. Unfortunately, the reproductive performance of Rarg epididymal sperm, their testes contain numerous Sertoli cell-
germ cell KO males has not been reported; based on the only tubules (lacking germ cells) adjacent to rather normal-
histology images provided in Gely-Pernot et al. [92], it is appearing tubules [116, 117]. A role in SSC self-renewal is
reasonable to expect that young KO mice are fertile. Therefore, concluded based on the histological phenotype and because
it can be concluded that although RAR isotypes clearly mutant spermatogonia are unable to colonize testes of recipient
participate in spermatogonial differentiation, none is essential mice lacking germ cells [116, 117].
for the process to occur. As mentioned above, seminiferous Without dramatic upregulation of mRNAs during spermato-
epithelial stages VII–VIII are exposed to the highest measured gonial differentiation, scientists have lacked targets in the form
RA levels [22, 43, 51], and spermatogonia differentiate during of proteins and pathways for focused studies. The lack of
this time. However, stage-VII and -VIII tubules also contain dramatic differences in the transcriptome of developing
SSCs, which remain undifferentiated and STRA8/KIT, spermatogonia suggests, first, very few changes in transcription
implying that they did not receive the RA signal. A recent or mRNA decay (both contribute to resulting steady-state
study employed a transgenic approach to create mice mRNA levels) or, second, differences in the transcriptomes
expressing RARG from the Gfra1 promoter in A within spermatogonial subtypes that are not discernible when
undiff
spermatogonia; the results indicate that these spermatogonia an entire population of spermatogonia is queried. Results from
inappropriately differentiate during stages VII–VIII [102]. a recent study suggest that both are viable options. Significant
These results, taken together with the Rarg KO data above, differences exist in the abundance of specific mRNAs within
support the concept that RARG is sufficient, but not required, single spermatogonia that correlate with their fate status
to direct spermatogonial differentiation. (expression of Id4-GFP in SSCs) [118]. This work also
The testis contains a consistently small SSC population and highlights examples of genes for which the mRNAs are present
a system for the proliferation and then differentiation of in Id4-GFPώ SSCs without detectable protein, which indicates
millions of progenitor spermatogonia required for gamete posttranscriptional regulation of gene expression.
production. Accordingly, it should not be surprising that the It has been known for many years that not all mRNAs are
responsiveness of spermatogonia to RA is controlled by translated into protein with similar efficiency—mRNAs can be
redundant mechanisms (expression of RARG in spermatogonia stored, can be inefficiently or efficiently translated, or can be
primed to differentiate but not in SSCs, and careful modulation targeted for degradation [119]. The decision among these
of RA levels by RA-synthesizing and -degrading enzymes) (see biochemical fates provides cells with an important level of
Fig. 3B). The evidence that such a robust system is seemingly control over gene expression that allows rapid and large-scale
not reliant upon the function of a single gene product highlights responses to developmental stimuli. Recent results provide a
how critical modulating male germ cell RA exposure is for novel perspective that may advance our understanding of
both male reproductive health and success. events during spermatogonial differentiation. Others and we
have reported that mRNAs encoding essential differentiation
GENE EXPRESSION CHANGES DURING DIFFERENTIA- factors (e.g., KIT, SOHLH1, and SOHLH2) are present, but
TION repressed, in A spermatogonia but then initiate translationundiff
in response to RA in A without a dramatic increase in
diff
Although it has been known for 90 yr that retinoids are mRNA abundance [42, 58, 120, 121]. This suggests that,
essential for male fertility [78], the molecular and cellular instead of transcriptional activation of unique genes, efficient
events downstream of RA remain largely undefined. A primary translation of a pool of repressed mRNAs is the driving force
reason for our lack of knowledge about spermatogonial behind gene expression changes during spermatogonial
differentiation is that studies using whole-genome approaches differentiation. Two well-studied mechanisms for posttran-
have identified few changes in steady-state mRNA levels scriptional control of gene expression involve miRNAs and
between A and A spermatogonia or in whole testes
undiff diff RNA-binding proteins, and evidence is growing that both are
during the early phases of neonatal testis development during involved in spermatogenesis.
which differentiation takes place [98, 106, 107]. Many of the MicroRNAs are short, noncoding RNAs that bind to target
upregulated spermatogonia-expressed genes (at the mRNA mRNAs and repress their translation by inducing cleavage or
level) encode proteins with known roles in meiosis (e.g., destabilization or by preventing ribosomal association (for
REC8, STRA8, and SYCP3) [53, 97, 108–110]. This is not review, see [122]). The biogenesis of requires the ribonuclease
meant to conclude that transcriptional regulation is uninvolved Dicer1. Somewhat surprisingly, deletion of Dicer1 in fetal
in spermatogonial differentiation. Indeed, the deletion of the male germ cells does not cause noticeable defects until the
transcription factors Sohlh1, Sohlh2, and Sox3 blocks (in pachytene stage of meiosis [123–127], indicating that miRNA
Sohlh1 and Sohlh2 KO testes) or impairs (in Sox3 KO testes) function is not essential for spermatogonial development.
spermatogonial differentiation, although the defects in Sox3 However, recent studies indicate that specific miRNAs and
germ cell KO mice are more severe during the first wave of miRNA clusters (Mir146, Mir221/222, Mirc1, Mirc3, and
spermatogenesis and improve over time as the mice age [56, Mirlet7) do contribute to gene regulation in spermatogonia
111–114]. In addition, testis cords are apparently normal [121, 128–130]. In vivo, however, they likely play a
through at least P10, which argues against an absolute supplementary or supportive role in the translational control
requirement for SOX3 during spermatogonial differentiation. of mRNAs encoding factors that direct spermatogonial
It has become clear through genomic and genetic analyses development. In contrast to the dispensable function of
that genes required to maintain SSCs in vivo and in vitro are miRNAs in premeiotic male germ cells, numerous RNA-
lost in response to spermatogonial differentiation signals [14– binding proteins have been shown to be essential for fetal and
17, 115]. One example is Zbtb16/Plzf, which encodes a neonatal mammalian germ cell development and differentiation
putative transcriptional repressor that is detectable in A (e.g., NANOS2, NANOS3, DAZL, TIAR/TIAL1, PIWIL2/
undiff
spermatogonia in the adult but is present in all spermatogonia MILI, PIWIL4/MIWI2, and DDX4/VASA) [131–135]. The
in the neonatal and juvenile testis [44, 116, 117]. ZBTB16 is most well characterized RNA-binding protein is NANOS2,
not absolutely required for spermatogenesis; although adult which is required for the function and survival of both fetal
mutant and KO mice are infertile, with very low numbers of prospermatogonia and postnatal SSCs [50, 136]. It appears to
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BUSADA AND GEYER
FIG. 4. RA stimulates transcription and PI3K/AKT/mTOR kinase signaling in differentiating spermatogonia. RA transcriptionally activates genes required
for meiosis (e.g., Stra8 and Rec8) and enhances the translational efficiency of repressed mRNAs required for spermatogonial differentiation (e.g., Kit,
Sohlh1, and Sohlh2) through activation of kinase signaling.
have multiple roles in suppressing translation of target mRNAs isoform plays during spermatogonial proliferation and differ-
during spermatogenesis, both by promoting their degradation entiation is currently unknown, but it is tempting to speculate
[137] and by preventing association with polyribosomes [138]. that it is required for the program of translational activation of
A formal link between RA and expression of NANOS2 has not repressed mRNAs, as described above.
been established in the postnatal testis, but in fetal testes of A perplexing question is why would undifferentiated male
mice lacking Cyp26b1 (which have higher RA levels), Nanos2 germ cells rely on a system of translational control over a
mRNA levels were significantly reduced [139]. This suggests subset of mRNAs, as it seems to be a rather inefficient use of
that RA may negatively regulate the expression of Nanos2; cellular resources? This difficult question has many potential
indeed, exogenous RA decreases Nanos2 mRNA levels in the answers, and we may never know which one is the most
neonatal testis (our unpublished data). The loss of NANOS2 correct. First, transcription is not a particularly expensive
expression downstream of RA signaling provides a potential process in terms of cellular ATP output, especially in
mechanistic explanation for the increased translational effi- comparison with protein synthesis and degradation [153,
ciency of repressed mRNAs encoding determinants of 154]. The simplest explanation may be that prospermatogonia
spermatogonial differentiation such as KIT, SOHLH1, and and A spermatogonia lack the ability to precisely control
undiff
SOHLH2. transcription of certain genes and therefore employ posttran-
Additional evidence supporting the importance of regulated scriptional controls as a means to regulate gene expression. An
protein synthesis during spermatogonial development comes example is provided by the KIT receptor tyrosine kinase;
from studies of mutant and KO mice. The first is from studies whereas the mRNA is detectable throughout testis develop-
using ‘‘juvenile spermatogonial depletion’’ (jsd) mutant mice, ment, the protein is only present in discrete stages (for review,
which exhibit a normal first wave of spermatogenesis followed see [52]). Protein is expressed in primordial germ cells and is
by a complete loss of all germ cells in the adult except for required for their proper migration to the developing fetal
A spermatogonia [140–143]. The mutated gene responsi-
undiff gonad. After their colonization, KIT protein expression is
ble for this phenotype was later identified as Utp14b [141, silenced for over a week in prospermatogonia but is required
142], which is an autosomally encoded, processed retroposon again beginning at P3–P4 in a subset of type A spermatogonia
copy of the X-linked Utp14a gene required for 18S ribosomal for their differentiation [52].
RNA processing during ribosome biogenesis. Interestingly,
raising testicular temperature in adult mice restores spermato- KINASE SIGNALING DURING SPERMATOGONIAL
genesis, leading to the creation of fertilizable sperm [144–146]. DIFFERENTIATION
This suggests that this naturally occurring mutation is
temperature-sensitive, which may explain why no phenotype The best-studied action of RA is genomic, in which RA
is apparent during the first wave of spermatogenesis, much of stimulates transcription by binding RARs on RA response
which occurs at 378C. The phenotype of adult mice, which lack elements (RAREs) in target gene promoters. However, lack of
UTP14B function, suggests that ribosome biogenesis is an significant changes in steady-state mRNA levels after RA
important aspect of spermatogonial proliferation and differen- exposure suggests other avenues of RA-based regulation may
tiation but is not critical in A spermatogonia. The second be utilized in the neonatal testis [98, 107]. Compelling
undiff
example is provided by mice lacking Eif2s3y, a Y-linked gene evidence in other systems indicates that RA can also utilize
that encodes a subunit of the eukaryotic initiation factor alternative, nongenomic pathways via kinase cascades [155,
complex EIF2, which forms a ternary complex with GTP and 156]. For example, RARA is bound to the regulatory subunit
Met-tRNA during translation initiation. KO testes only contain (p85) of PI3K in several cell types (SH-SY5Y, NIH3T3, and
GFRA1ώ A spermatogonia, indicating that EIF2S3Y is MEFs). RA addition causes the recruitment of the catalytic
undiff
required for spermatogonial proliferation and differentiation subunit (p110) to induce rapid phosphorylation of ERK and
[147–151]. Coincidentally, Eif2s3y is one of the two genes (the AKT [155, 156]. In support of this, our laboratory discovered
other being Sry) on the Y chromosome required for male that RA activates the PI3K/PDK1/AKT/mTORC1 signaling
fertility [152]. What specific role this EIF2 gamma subunit pathway, and this is required for translation of repressed
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SPERMATOGONIAL DIFFERENTIATION IN THE MOUSE
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