small rnas in spermatogenesis
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Small RNAs in spermatogenesis
Ram Prakash Yadav, Noora Kotaja
PII: S0303-7207(13)00160-3
DOI: http://dx.doi.org/10.1016/j.mce.2013.04.015
Reference: MCE 8498
To appear in: Molecular and Cellular Endocrinology Molecular
and Cellular Endocrinology
Please cite this article as: Yadav, R.P., Kotaja, N., Small RNAs in spermatogenesis, Molecular and Cellular
Endocrinology Molecular and Cellular Endocrinology (2013), doi: http://dx.doi.org/10.1016/j.mce.2013.04.015
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Small RNAs in spermatogenesis
Ram Prakash Yadava and Noora Kotajaa,*
a Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10,
FIN-20520 Turku, Finland, [email protected], [email protected]
*Corresponding author: Noora Kotaja
Department of Physiology
Institute of Biomedicine
University of Turku
Kiinamyllynkatu 10
FIN-20520 Turku, Finland
Tel: +358-2-3337283
E-mail: [email protected]
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Abstract
Spermatogenesis is characterized by meiotic divisions and major morphological changes to
produce spermatozoa that are capable of independent movement and fertilization of an egg.
Male germ cell differentiation is governed by orchestrated, phase-specific gene expression
patterns that are tightly controlled at transcriptional and post-transcriptional level. Post-
transcriptional regulation of protein-coding mRNAs becomes prominent during the late steps
of spermatogenesis when the compacting sperm nucleus becomes transcriptionally inhibited.
Small non-coding RNAs are important regulators of gene expression that mainly function
post-transcriptionally to control the properties of their target mRNAs. Male germ cells express
several classes of small RNAs, including Dicer-dependent microRNAs (miRNAs) and
endogenous small interfering RNAs (endo-siRNAs), as well as Dicer-independent piwi-
interacting RNAs (piRNAs). Increasing evidence supports the essential role of small RNA-
mediated RNA regulation in normal spermatogenesis and male fertility.
Keywords Spermatogenesis; Germ cell; Fertility; Post-transcriptional; Non-coding RNA; Small RNA
Abbreviations PGC, primordial germ cell; CB, chromatoid body; miRNA, microRNA; siRNA, small interfering
RNA; endo-siRNA, endogenous small interfering RNAs; piRNA, PIWI-interacting RNA; RISC,
RNA-induced silencing complex; MSCI, meiotic sex chromosome inactivation; SSC,
spermatogonial stem cell; RNP, ribonucleoprotein; AGO, Argonaute protein; PND, post-natal
day; TE, transposable element;
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1. Introduction to spermatogenesis Development of male gamete is a complex differentiation program that produces highly
specialized spermatozoa (Hess and Renato de Franca, 2008). Germ cells are unique in their
capability to generate new organisms, and therefore molecular events during
spermatogenesis have to be strictly regulated to enable correct transmission of genetic and
epigenetic information to subsequent generations. Spermatogenesis is an efficient process
and average healthy human male may produce as much as thousand spermatozoa each
second. Spermatogenesis is also prone to mistakes as demonstrated by the low percentage
of morphologically normal spermatozoa in an average human semen sample. Moreover,
alarming adverse trends in the semen quality in Western countries have been reported
(Sharpe, 2012). At the same time, incidence of testicular germ cell tumors and congenital
malformations of the reproductive organs is increasing, which highlights the importance of
the characterization of the environmental and genetic factors negatively affecting male
reproductive health.
Male germ cells are specified shortly after implantation from the pluripotent cells of epiblast.
During gastrulation, the nascent primordial germ cells (PGCs) start to migrate toward the
future gonads and rapidly proliferate (Matsui, 2010). After migrating to the gonadal ridge,
PGCs become gonocytes within cords that are formed by Sertoli precursors and surrounded
by peritubular cells. Gonocytes show a burst of mitotic activity, then arrest in the G0 phase of
cell cycle remaining mitotically quiescent until after birth, when they give rise to
spermatogonia (de Rooij and Russell, 2000). Embryonic germ cells undergo a genome wide
epigenetic reprogramming, which is initiated by near to complete erasure of somatic
epigenetic marks. Subsequently, novel sex-specific DNA methylation patterns, including
differential imprinting of the genes in the male and female germ cells, are established (Sasaki
and Matsui, 2008; Ewen and Koopman, 2010).
Spermatogenesis starts shortly after birth by mitotic proliferation of spermatogonia (Fig. 1).
After proliferation phase, spermatogonia enter the meiotic phase and become
spermatocytes. After the long-lasting meiotic prophase I that includes homologous
chromosome pairing, synapse and recombination, cells undergo a reduction division during
which the sister chromosomes are separated into two cells to generate secondary
spermatocytes. These cells very quickly divide again to give raise to haploid round
spermatids. All the dramatic morphological changes such as acrosome and flagellum
formation, nuclear shaping and chromatin condensation take place during the post-meiotic
spermatid differentiation (spermiogenesis). Chromatin is tightly compacted with the help of
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sperm-specific protamines that replace the most of histones and enable more efficient
packing of DNA. As a result of spermiogenesis, the spermatozoa are equipped with all the
structures required for its prospective functions. Spermatozoa are released into a lumen of
seminiferous tubules, where they continue the journey to the epididymis. Transit of
spermatozoa through the long convoluted epididymal duct is important for their final
maturation and acquirement of the capacity for full motility.
Germ line stem cells located in the basal compartment of seminiferous epithelium provide a
source of undifferentiated cells that enable the production of spermatozoa throughout the
whole period of sexual maturity (de Rooij and Russell, 2000). Interestingly, recent studies
have succeeded to generate totipotent embryonic stem cells from male germ line stem cells
(Fagoonee et al., 2011). Therefore, male germ line stem cells isolated from the adult testis
may offer a powerful tool for regenerative medicine. The seminiferous epithelium is organized
in cyclic stages so that each cross-section of the tubule contains a defined grouping of germ
cell types at particular phases of development. In mice, there are twelve designated cell
associations or stages (I-XII) that are arranged in an ordered manner in the logical sequence
of developmental progression (Kotaja et al., 2004). This organization ensures the production
of a constant supply of mature spermatozoa. It also enables the coordinated regulation of
germ cells at different phases of differentiation by the surrounding Sertoli cells that embed
germ cells in the cytoplasmic pockets. Spermatogenesis is fully dependent on close
interactions with Sertoli cells that provide physical and nutritional support to germ cells (Rato
et al., 2012).
Male reproductive functions are controlled by the synchronized action of the hypothalamic-
pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) from hypothalamus
regulates the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from
anterior pituitary (Ruwanpura et al., 2010). FSH and LH are targeted to the testis where they
mostly act on somatic testicular cell types to stimulate testosterone production by interstitial
Leydig cells or to regulate Sertoli cells. Testosterone is absolutely required for maintaining
spermatogenesis, the main targets being meiosis and spermiogenesis (Ruwanpura et al.,
2010). In addition to the extrinsic hormonal and paracrine control, a wide variety of germline-
specific intrinsic delicate regulatory mechanisms are required. In this review, we concentrate
in describing the post-transcriptional gene control in male germ cells and summarizing the
results reporting the emerging importance of small non-coding RNAs in spermatogenesis and
male fertility.
2. Post-transcriptional gene regulation in spermatogenesis
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The accurate, spatially and temporally regulated gene expression is of fundamental
importance for normal progression of spermatogenesis. Regulation takes place at
transcriptional and epigenetic levels, but in addition, post-transcriptional gene control has an
essential role especially at later steps of sperm differentiation when chromatin compaction
induces transcriptional silencing in elongating spermatids (Kimmins et al., 2004; Kimmins
and Sassone-Corsi, 2005). mRNAs for many spermiogenic proteins are synthesized already
in meiotic cells, and are temporarily stored and translationally regulated until needed during
later steps. As an example, mRNAs for protamines are kept inactive for several days until the
proteins are needed for histone-protamine transition, and premature translation leads to
spermatogenic defects (Lee et al., 1995). Fates of mRNAs are controlled mainly by RNA-
binding proteins that bind their targets either in a non-specific manner or through specific
motifs. There are a wide range of different RNA-binding proteins, also testis-specific ones,
expressed in meiotic and post-meiotic cells that participate in mRNA regulation by
recognizing target mRNAs and forming ribonucleoprotein (RNP) complexes (Paronetto and
Sette, 2010; Idler and Yan, 2012).
Cytoplasmic RNP granules provide important platforms for the post-transcriptional RNA
control (Meikar et al., 2011). For example processing bodies (P-bodies, PBs or GW/P bodies)
in mammalian cultured cells are known to coordinate mRNA decay, storage, transport and
miRNA-mediated pathways (Kulkarni et al., 2010). Germ cells of various organisms are
characterized by special cytoplasmic RNP granules that are commonly called germ granules.
The biological functions of the germ granules in non-mammalian and mammalian organisms
appear to be distinct, but they still share many protein components and are all involved in the
RNA regulation (Meikar et al., 2011; Kotaja and Sassone-Corsi, 2007; Chuma et al., 2009).
Germ granules exist in the cytoplasm of germ cells during the whole course of
spermatogenesis. The most prominent germ granules are intermitochondrial cement (IMC) in
spermatocytes that form between mitochondrial clusters, and the chromatoid body (CB) in
haploid round spermatids. These two types of granules share many protein components that
are mainly RNA-binding proteins, RNA helicases, Tudor domain proteins, and other proteins
involved in RNA processing. The germ granules seem to be central structures in the
processes involving PIWI-interacting RNA (piRNA) pathways (Meikar et al., 2011). The CB is
the biggest of all the known RNP granules. In mouse, the CB appears in pachytene
spermatocytes as a fibrous–granular structure and condenses into one single finely
filamentous, lobulated, perinuclear granule in post-meiotic round spermatids (Meikar et al.,
2011; Kotaja and Sassone-Corsi, 2007). The important role of the CB in male reproduction is
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highlighted by the infertile phenotype of many knockout mice lacking various CB components
(Kotaja and Sassone-Corsi, 2007).
Recent evidence demonstrates that most of the human genome is transcribed but only a
small fraction of the transcribed RNA encodes for proteins (Rinn and Chang, 2012; Guttman
and Rinn, 2012). Therefore, most of the RNA transcripts are non-coding, i.e. they are not
used as a template for protein synthesis. It has become clear that these non-coding RNAs
are not just “junk” as it was originally thought, but they serve important cellular functions. In
addition to ribosomal RNA, transfer RNA, small nucleolar RNA (snoRNA), small nuclear RNA
(snRNA) and other well characterized functional RNAs, cells transcribe a legion of other
kinds of non-coding RNAs with different sizes and different functions. Non-coding RNAs have
been shown to have critical role in the control of gene expression, functioning both at
transcriptional level as components of chromatin remodeling complexes or post-
transcriptionally (Rinn and Chang, 2012). Male germ cells, especially meiotic cells, are
transcriptionally very active. Male germ cells express also an outstanding amount of different
non-coding RNAs, including both yet poorly characterized long non-coding RNAs and small
RNAs that are known to be essential for male germ cell-specific processes.
3. Control of gene expression by small RNAs Small non-coding RNAs are probably the best characterized class of non-coding RNAs in
terms of their functions. These RNAs do not code for proteins, however, they function as
cellular modulators of gene expression (Ghildiyal and Zamore, 2009; Ketting, 2011; Carthew
and Sontheimer, 2009). Discovery of microRNAs (miRNAs) started in Victor Ambros’
laboratory in 1993 while working on lin4 and how it controls developmental timing in the C.
elegans (Lee et al., 1993). Today after 20 years of their discovery, miRNAs are recognized as
vital regulatory factors that control the expression of a broad range of protein coding genes.
miRNAs function mainly post-transcriptionally by affecting the stability of their target mRNAs
or in some cases the translation of mRNAs (Ghildiyal and Zamore, 2009; Ketting, 2011;
Carthew and Sontheimer, 2009). In addition, transcriptional control of gene expression is
well-established in the plants (Khraiwesh et al., 2010) and also recently observed in human
granulopoiesis (Zardo et al., 2012).
miRNAs are synthesized as hairpin loop precursors and processed first by the nuclear
enzyme Drosha (Fig. 2) (Siomi and Siomi, 2010; Krol et al., 2010; Winter et al., 2009). After
the transport to the cytoplasm, they are further processed by the endonuclease Dicer into 21-
25 nucleotide double-stranded RNAs (dsRNAs). The produced dsRNA is unwound and one
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of the strands is used as template strand which in association with accessory proteins and
Argonaute (AGO) proteins form the RNA-induced silencing complex (RISC) (Pratt and
MacRae, 2009; Hutvagner and Simard, 2008). miRNAs mediate the sequence-specific
recognition of their target mRNAs by the RISC complex. Depending upon sequence
complementary, the target mRNA is either degraded or translationally silenced. The same
pathway including Dicer and RISC-directed cleavage is used in antiviral defense to
specifically target and inactivate invading nucleic acids. This RNA silencing mechanism
couple the destruction of viral RNA with the use of the resulting small interfering RNAs
(siRNAs) to target other nucleic acid molecules that contain the complementary sequence
(Mlotshwa et al., 2008).
Several miRNAs can target one mRNA. On the other hand, one miRNA can down-regulate
the expression level of hundreds of genes and act as a regulatory factor to control various
physiological processes and diseases (Sood et al., 2006). Mammalian genomes comprise
roughly a thousand miRNA genes. Tissue-specific expression patterns of miRNAs are
observed (Lagos-Quintana et al., 2002), and misregulation of miRNA expressions seems to
be a common finding in cancer cells. miRNA profiling is used for many cancer sub-
classifications and identifications of cancer tissue origin (Lu et al., 2005; Rosenfeld et al.,
2008). Due to the high requirements for gene regulation, it is not a surprise that small non-
coding RNAs are also involved in the control of sperm production. Male germ cells express
several classes of small RNAs, including Dicer-dependent miRNAs and endogenous siRNAs
(endo-siRNAs), as well as Dicer-independent PIWI-interacting RNAs (piRNAs) (Meikar et al.,
2011). These small RNAs and their roles in spermatogenesis will be discussed in the
following chapters.
4. Dicer-dependent small RNAs in male germ cells
4.1. miRNAs and endo-siRNAs
Post-transcriptional mRNA regulation is active during spermatogenesis, and miRNA-
mediated mechanisms are clearly involved in the orchestrated and stage-specific control of
gene expression during the whole course of male germ cell differentiation. Several studies
using miRNA microarrays, RT-PCR or small RNA sequencing have identified miRNAs that
are highly, exclusively or preferentially expressed in the testis and at the specific phases of
male germ cell differentiation (Papaioannou and Nef, 2010; McIver et al., 2012a). These
studies have demonstrated for example differentially expressed miRNAs between immature
and mature mouse testes (Yan et al., 2007), specific miRNA profiles in highly enriched
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populations of gonocytes and spermatogonia (McIver et al., 2012b), and miRNA profiles
during the first wave of spermatogenesis by using prepubertal testes collected on post-natal
days (PND) 7, 10 and 14 (Buchold et al., 2010). miRNA profiles were identified also from
enriched populations of stem cells, pre-meiotic and meiotic cells (Smorag et al., 2012) or
spermatogonia, spermatocytes and spermatids (Marcon et al., 2008). These cell types seem
to express several common miRNAs, but also some miRNAs that are specific for the certain
cell type.
Intriguingly, densities of miRNA genes are significantly higher on the mammalian X
chromosome than on autosomes as a consequence of the organization of X-chromosomal
miRNA genes in clusters that consist of paralogous copies resulting from miRNA gene
duplication (Meunier et al., 2013). Sequencing of spermatocyte and spermatid RNA in
comparison to somatic cells revealed that the multimember family miRNAs on the X have
higher expression levels than all other miRNA categories in male germ cells (Meunier et al.,
2013). Sex chromosomal genes are normally silenced by the process of meiotic sex
chromosome inactivation (MSCI) during meiotic prophase I. However, miRNAs and
especially the miRNA families that expanded by gene duplication, seems to escape silencing
by MSCI, suggesting that the miRNA gene duplications on the X was selectively favored
during evolution to allow their expression in spermatocytes and spermatids in spite of sex
chromosome inactivation (Meunier et al., 2013; Song et al., 2009).
The identification of functions and target genes for specific miRNAs during spermatogenesis
has already begun. Several studies support the role of miRNA-mediated gene control in the
maintenance of undifferentiated state of spermatogonia and the induction of differentiation.
miR-146 that is highly expressed in undifferentiated spermatogonia is involved in the control
of retinoid acid-induced spermatogonial differentiation in the mouse (Huszar and Payne,
2013). This is at least partially mediated by the regulation of the expression of the mediator
complex subunit 1 (Med1), a coregulator of retinoid receptors. Other miRNAs involved in the
regulation of the undifferentiated vs. differentiated state of spermatogonia are miR-221 and
miR-222. The impaired function of these X chromosome-clustered miRNAs in mouse induces
spermatogonial differentiation (Yang et al., 2013). Mir-17-92 and its paralog Mir-106b-25
clusters are also expressed in undifferentiated spermatogonia and down-regulated during
differentiation. Deletion of Mir-17-92 cluster in mouse results in small testes and lower
number of epididymal sperm and increase in Mir-106b-25 expression which suggests a
functional co-operation of these two clusters (Tong et al., 2012). High-throughput sequencing
identified miR-21, along with miR-34c, -182, -183, and -146a, as being preferentially
expressed spermatogonial stem cell (SSC)-enriched population. The transient inhibition of
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miR-21 in SSC-enriched germ cell cultures increased the number of germ cells undergoing
apoptosis and reduced the SSC potency, indicating that miR-21 is important for maintaining
the SSC population (Niu et al., 2011).
miR-34c is highly expressed in spermatocytes and round spermatids (Romero et al., 2011;
Bouhallier et al., 2010; Liang et al., 2012). Inhibition of miR-34c in primary spermatocytes
seems to prevent germ cell from testosterone deprivation-induced apoptosis, and
overexpression of miR-34c in cultured germ cells trigger apoptosis. This is at least partially
mediated by targeting ATF1 via direct binding to ATF1 3’UTR (Liang et al., 2012). When
overexpressed in HeLa cells, miR-34c induces a shift in the transcriptome towards the germ
lineage transcriptome (Bouhallier et al., 2010). Therefore, miR-34c was suggested to play a
role in enhancing the germinal phenotype of cells already committed to this lineage.
Interestingly, miR-34c is also found in mature sperm and the zygotes, and the sperm-borne
miR-34c appears to be important for the first zygotic cell division via modulation of Bcl-2
expression (Liu et al., 2012b). miR-34c is not an only example of sperm-derived miRNAs.
Despite the transcriptionally inactive state of mature spermatozoa, its nucleus contains a
complex population of both mRNAs and miRNAs that may have a role in early embryo
development, and that can be potentially exploited in the investigation and diagnostics of
male infertility (McIver et al., 2012a; Hamatani, 2012; Abu-Halima et al., 2013).
Other miRNAs involved in the regulation of meiotic and post-meiotic gene expression have
been reported. miR-449 cluster miRNAs are drastically up-regulated upon meiotic initiation
during first wave of spermatogenesis, and their expression is under the regulation of
transcription factors CREMtau and SOX5 (Bao et al., 2012). miR-449 cluster and miR-34b/c
were shown to share some target genes that belong to the E2F transcription factor-
retinoblasoma regulatory network (Bao et al., 2012). The correct timing of the expression of
transition proteins (TP) and Protamines (Prm) is critical for successful histone-protamine
transition during late spermatogenesis. TPs and Prms are subjected to extensive
translational control, including miRNA-mediated mechanisms. TP2 and Prm2 mRNAs are
targeted by the testis-specific miR-469 that represses TP2 and Prm2 protein expression in
pachytene spermatocytes and round spermatids at the translation level with minor effect on
mRNA degradation (Dai et al., 2011). miR-122a that is enriched in late-stage male germ cells
can also directly control TP2 expression by inducing mRNA cleavage (Yu et al., 2005). miR-
18 that belongs to the Oncomir-1 or miR-17-92 cluster can directly target heat shock factor 2
(HSF2), a transcription factor that influences a wide range of developmental processes
including spermatogenesis (Bjork et al., 2010). The expressions of HSF2 and miR-18 exhibit
an inverse correlation during spermatogenesis and inhibition of miR-18 in intact seminiferous
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tubules leads to increased HSF2 protein levels and altered expression of HSF2 target genes
(Bjork et al., 2010).
Recently, endogenous siRNAs (endo-siRNAs) which are processed by Dicer were reported
to be highly expressed in mouse male germ cells (Song et al., 2011). Endo-siRNAs do not
need the microprocessor complex (Drosha-DGCR8) for their biogenesis because they are
processed from long dsRNA precursors; nuclear Drosha activity is required only for the
processing of the stem-loop structure-containing miRNA precursors (Krol et al., 2010). Endo-
siRNAs were initially identified in yeasts, plants, C. elegans and Drosophila (Czech et al.,
2008; Okamura et al., 2008; Chapman and Carrington, 2007). In the mouse, they have been
reported in embryonic stem cells (Babiarz et al., 2008) and oocytes (Watanabe et al., 2008;
Tam et al., 2008) in addition to the testis. A total of 73 endo-siRNAs were identified by
sequencing the testicular RNA. The majority of them were mapped to several (even
hundreds) of different sites on multiple chromosomes in contrast to miRNAs that usually
derive from a unique locus or very few loci (Song et al., 2011). Predicted targets for the
testicular endo-siRNAs were mainly mRNAs (~92%) but also transcripts of pseudogenes
(~3%), retrotransposons (~1%), and non-coding RNAs (~4%). In mouse oocytes, endo-
siRNAs seem to regulate both retrotransposons and protein-coding transcripts (Watanabe et
al., 2008; Tam et al., 2008). Testicular endo-siRNAs are probably involved in post-
transcriptional RNA regulation since they effectively induced target mRNA degradation in
vitro. Since they display numerous hits on multiple chromosomes, it is tempting to speculate
that in addition to their role in post-transcriptional RNA control, they could also have nuclear
effects on chromatin modifications similar to what has been shown for endo-siRNAs in other
organisms (Song et al., 2011).
4.2. Male germ cell-specific Dicer and Drosha knockout mouse models
miRNA and siRNA processing is globally dependent on the two endonucleases, the
microprocessor complex (Drosha/DGCR8) and Dicer. Both Dicer and DGCR8 have been
demonstrated to be essential genes in mouse since their deletion leads to an embryonic
lethal phenotype (Bernstein et al., 2003; Wang et al., 2007). Tissue and cell type–specific
knockout mouse models have enabled the assessment of the functions of these pathways in
certain tissues and in adult organism. By using this approach, the role of miRNA/siRNA
pathways in spermatogenesis has started to be revealed (Fig. 1, Table 1). Dicer has been
demonstrated to be required for the functions of both Sertoli cells that are the supporting
somatic cells of the seminiferous epithelium (Papaioannou et al., 2009; Papaioannou et al.,
2011) and germ cells that will be discussed in more detail. Removal of Dicer1 gene in
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primordial germ cells after embryonic day 10 (E10) by using the tissue-nonspecific alkaline
phosphatase (Tnap) promoter-driven Cre expression demonstrated for the first time the
importance of Dicer for male fertility (Hayashi et al., 2008; Maatouk et al., 2008). Upon
removal of functional Dicer, the PGCs showed defects in proliferation and post-natal
spermatogenesis was also affected (Hayashi et al., 2008; Maatouk et al., 2008). This mouse
model could not be used to address the exact role of Dicer in adult spermatogenesis due to
affected embryonic germ line development, and the low penetrance of TnapCre transgene
which interferes with the analysis.
Conditional deletion of Dicer1 in the male germ line just before birth by using the Ddx4
promoter-driven Cre expression resulted in severe cumulative defects in meiotic and post-
meiotic germ cells (Romero et al., 2011; Liu et al., 2012a). The spermatocytes were delayed
in the progression of meiotic prophase I and apoptosis was increased, resulting in reduced
number of round spermatids. The remaining round spermatids failed to undergo normal
differentiation and morphologically abnormal non-functional spermatozoa were generated
(Romero et al., 2011). The expression of transposable elements of the SINE (short
interspersed nuclear element) family was up-regulated in Dicer1 knockout spermatocytes
(Romero et al., 2011). Similar up-regulation of transposon expression (MT and SINE families)
was observed in Dicer1 deficient mouse oocytes (Murchison et al., 2007). Dicer function has
also been linked to transposon silencing in retinal pigmented epithelium where it is involved
in retrotransposon transcript degradation by a miRNA-independent mechanism (Kaneko et
al., 2011).
Interestingly, less severe phenotypes were observed when Dicer1 was deleted after birth in
post-natal spermatogonia in Neurogenin3 (Ngn3)-Cre and Stra8Cre knockout models
(Korhonen et al., 2011; Greenlee et al., 2012; Wu et al., 2012). Selective removal of Dicer1 in
type A spermatogonia by Ngn3Cre transgene did not have clear effects on meiotic
progression and the first visible impairments were found in haploid male germ cells
(Korhonen et al., 2011). Elongation phase of spermiogenesis was severely affected with
problems in chromatin organization and shaping and condensation of sperm head (Korhonen
et al., 2011). The number of epididymal spermatozoa was dramatically reduced and all
spermatozoa were morphologically abnormal. Interestingly, transposon expression was
unaffected in the knockout testis. The difference in transposon expression between the
Ddx4Cre and Ngn3Cre–induced Dicer1 knockout models indicate that Dicer may be involved
in transposon control in embryonic and/or very early post-natal phases before the onset
Ngn3 expression. On the other hand, major satellite expression was induced in the testis of
Ngn3Cre-Dicer1 knockout mice similarly to what is observed in mouse embryonic stem cells
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lacking functional Dicer (Korhonen et al., 2011; Murchison et al., 2005; Kanellopoulou et al.,
2005). Altogether the reported defects in repeat sequence expressions (transposon and
satellite repeats) in different Dicer knockout models suggest that Dicer is implicated in the
control of repetitive elements in addition to its essential role in miRNA biogenesis.
The induction of Dicer1 deletion in early spermatogonia by Stra8Cre transgene resulted in
the comparable phenotype to Ngn3Cre driven Dicer1 deletion (Greenlee et al., 2012; Wu et
al., 2012). The meiotic progression of Stra8Cre-Dicer1 knockout spermatocytes was delayed
and haploid differentiation was greatly affected. The microarray analysis of PND18 testes of
the control and knockout mice revealed that ~40% of the genes were differentially expressed
in the knockout testis (Greenlee et al., 2012). Interestingly, disproportionately high
percentage of X- and Y-linked genes was overexpressed in the knockout testis. However, it is
still unclear if the overexpression of the genes that should be silenced during MSCI is
induced by direct defects in MSCI or if it originates from the increased transcript stability or
by the enrichment of pre-MSCI spermatocytes in the analysed knockout testis samples
(Greenlee et al., 2012). The ablation of Dicer or Drosha in spermatogonia did not appear to
affect significantly the sex body formation in pachytene spermatocytes (Wu et al., 2012). It is
evident that the mechanisms of Dicer-mediated sex chromosomal gene silencing require
further investigations.
Male germ cell–specific Dicer1 knockout mouse has also been generated by using protamine
1 (Prm1)-Cre transgene, that is activated much later in spermatogenesis in haploid cells
(Chang et al., 2012). The spermatogenesis of these mice was not so severely affected as the
spermatogenesis in Dicer1 knockout models in which the deletion takes place already in
spermatogonia (Korhonen et al., 2011; Greenlee et al., 2012; Wu et al., 2012). In
consequence, a higher number of spermatozoa were also found in the epididymides of
Prm1Cre-Dicer1 knockout males. However, post-meiotic differentiation was disrupted and
elongating spermatids had abnormal head morphology and compromised chromatin integrity
(Chang et al., 2012). Dicer1 inactivation in round spermatids impaired translational activation
of germ cell transcripts including Prm1, possibly resulting from the transcript sequestration
into translationally inert RNP complexes. All these different Dicer1 knockout models jointly
suggest that Dicer activities are required throughout the male germ cell differentiation, and
the earlier the deletion takes place, the more defects accumulate and the more compromised
the spermatogenesis is.
Since Dicer is required for the processing of both miRNAs and endo-siRNAs, it is important
to dissect these pathways and clarify the involvement of specific RNA species in the
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regulation of spermatogenic processes. The functional difference between miRNA and endo-
siRNA pathways in spermatogenesis has been investigated by comparing the testicular
phenotypes of Dicer1 and Drosha knockout mice (deletion induced in spermatogonia by
Stra8Cre transgene) (Wu et al., 2012). Drosha knockout testis that has defective miRNA
pathway but intact endo-siRNA pathways displayed more severe spermatogenic disruptions,
thus highlighting the essential role of miRNA pathways in normal post-natal
spermatogenesis. This is in contrast to female germ cells that appear to be more dependent
on endo-siRNA pathways; mRNA profiles of wild-type and Dgcr8 null oocytes were identical,
whereas Dicer null oocytes depicted many misregulated transcripts, therefore suggesting
that endo-siRNAs are important during oocyte maturation and preimplantation development
(Suh et al., 2010). The transcriptome analysis revealed that mRNA profiles of Drosha- or
Dicer-null pachytene spermatocytes and round spermatids were altered (Wu et al., 2012).
Even though some mRNAs showed similar changes in both mouse lines, many changes in
the mRNA expressions were unique to either of the two genotypes. This indicates that
miRNAs and other Dicer-dependent pathways (e.g. endo-siRNAs) have divergent roles in the
regulation of protein-coding mRNAs during post-natal spermatogenesis (Wu et al., 2012).
The roles of small RNA pathways can be also studied by inactivating the effector proteins
that mediate miRNA/siRNA functions. AGO proteins (AGO1-AGO4) associate with miRNAs
and siRNAs and mediate their effects through formation of RISC complexes. AGO proteins
are ubiquitously expressed, but AGO4 is highly expressed in testis and interestingly, it
localizes to the transcriptionally silenced sex body during the meiotic prophase I
(Modzelewski et al., 2012). Ago4 knockout spermatogonia initiate meiosis early and manifest
incorrect sex body assembly, which leads to disrupted MSCI. Interestingly, AGO4 deletion is
accompanied by the decreased expression of X-chromosomal miRNAs known to escape
MSCI (Song et al., 2009; Modzelewski et al., 2012). These findings clearly demonstrate the
role of AGO4 in the nucleus of pachytene spermatocytes in regulating MSCI, and implicates
that small RNA pathways are involved in this process. Some miRNAs localize in the nucleus
of pachytene spermatocytes and in the sex body further supporting the role of miRNA
pathway in MSCI (Marcon et al., 2008). Sex chromosome regulation is clearly a central target
for Dicer and small RNAs, but the exact relationship between Dicer, miRNAs, AGO4 and
MSCI remains to be explored.
5. PIWI proteins and piRNAs
piRNAs form a big and complex group of small RNAs that were discovered in 2006 by high-
throughput sequencing of either total testis RNA or RNA species interacting with PIWI
14
proteins (Aravin et al., 2006; Girard et al., 2006; Grivna et al., 2006; Watanabe et al., 2006).
piRNAs are 24-30 nucleotides, single-stranded RNAs that are synthesized in very high
numbers by Dicer-independent biogenesis mechanism and that are expressed predominantly
in the male germ line (Chuma and Nakano, 2013). piRNA encoding genes are mostly found
in genomic clusters ranging from a few to hundreds of kilobases in length. piRNAs in the
clusters map typically to only one genome strand of each cluster. This kind of asymmetric
organization suggests that long single-stranded precursor piRNA transcripts are transcribed
form the piRNA clusters. piRNA biogenesis from long transcripts requires at least two
nucleolytic steps to process both the monophosphorylated 5´end and the 3´end. The details
of the primary piRNA biogenesis are still obscure, but recently a protein
PLD6/MITOPLD/Zucchini was suggested to act in primary piRNA biogenesis as single-
strand-specific nuclease (Nishimasu et al., 2012; Ipsaro et al., 2012). To support this function,
primary piRNA generation was demonstrated to be defective in Pld6 knockout mice
(Watanabe et al., 2011; Huang et al., 2011). In mammals, piRNAs are classified mainly into
three groups on the basis of their stage-specific expression patterns: fetal piRNAs and post-
natal pre-pachytene and pachytene piRNAs (Fig. 1) (Chuma and Nakano, 2013). Sequence
comparison shows that these piRNA populations expressed at different developmental
stages are quite distinct and they originate from chromosomal clusters showing little overlap.
piRNA sequences are not conserved between the species but the locations of piRNA clusters
seems to be conserved. This suggests the involvement of chromatin-based regulatory
mechanisms in the control of piRNA cluster expression, even though nothing is known so far
about the transcriptional or developmental regulation of the piRNA clusters.
5.1. piRNAs and genome defense
To date, piRNAs in mammals and lower organisms have been demonstrated to be involved
in male germ line development, epigenetic regulation and mobile element repression (Chuma
and Nakano, 2013). Especially the role of piRNA machinery in genome defense and
transposable element (TE) control has been convincingly demonstrated in fruit fly, worm and
fetal testis of the mouse (Aravin et al., 2008; Kuramochi-Miyagawa et al., 2008; Siomi et al.,
2011; Bao and Yan, 2012). TEs are mobile DNA segment that can move and replicate within
the host genome and if aberrantly expressed, can result in potentially harmful insertional
mutagenesis and genomic rearrangements. TE expression is controlled at transcriptional
level by DNA methylation and heterochromatin formation, and post-transcriptionally by small
RNAs. Male germ cells are at high risk of transposon attack during the time of epigenetic
reprogramming in the fetal testis which in turn leads to temporary relaxation of TEs from
epigenetic control. Therefore, an adaptive strategy has evolved in germ cells to silence TEs
15
and to maintain genomic integrity. piRNA machinery seems to contribute to this genome
defense in a significant way. piRNA-mediated transposon silencing takes place at the post-
transcriptional level to cleave transposon transcripts, but a connection between piRNA action
and DNA methylation of transposon genes has also been demonstrated (Aravin et al., 2008;
Kuramochi-Miyagawa et al., 2008). A specific mechanism, so-called ping-pong amplification
cycle, is utilized to amplify piRNAs during TE control (Fig. 3). In this pathway, primary piRNAs
loaded to PIWI proteins recognize complementary target RNAs that are cleaved to produce
secondary piRNAs that in turn continues the cycle to amplify piRNA response.
piRNA functions are mediated through binding to PIWI proteins. There are three PIWI
subfamily members in the mouse, PIWIL1/MIWI, PIWIL2/MILI and PIWIL4/MIWI2 that are
expressed solely in the germ line with distinct expression patterns (Deng and Lin, 2002;
Kuramochi-Miyagawa et al., 2004; Carmell et al., 2007). MIWI2 has a very narrow expression
window from embryonic prospermatogonia until the very early post-natal spermatogonia,
corresponding to the time of epigenetic reprogramming and de novo DNA methylation
(Carmell et al., 2007; Aravin et al., 2009). In these cells, MIWI2 localizes in the nucleus and
in cytoplasmic granules (piP-bodies) that are also positive for MEAL, DDX4, TDRD9 and
canonical Processing body (P-body) proteins (Aravin et al., 2009). MILI co-exists with MIWI2
in prospermatogonia but it localizes in another kind of germ granules, pi-bodies, together with
TDRD1 and DDX4. These two types of germ granules are in close connection and participate
in piRNA biogenesis, de novo DNA methylation and transposon silencing (Aravin et al.,
2009). Knockout mouse models for Miwi2 or Mili genes generate a testicular phenotype with
spermatogenesis arrested at early meiotic stage (Kuramochi-Miyagawa et al., 2004; Carmell
et al., 2007) possibly resulting from the genomic instability due to an aberrant TE
expressions.
5.2. piRNAs in post-natal spermatogenesis
Two of three PIWI proteins are expressed during post-natal spermatogenesis where the
functions of piRNAs are much less understood than in prospermatogonia. MILI is found in the
cytoplasm of spermatogonia, pachytene spermatocytes and round spermatids, whereas
MIWI expression starts in meiotic cells and continues during the whole course of round
spermatid differentiation (Chuma and Nakano, 2013). In meiotic and post-meiotic cells, MIWI
and MILI predominantly localize in germ granules such as IMC in spermatocytes and CBs in
spermatids (Meikar et al., 2011; Deng and Lin, 2002; Kuramochi-Miyagawa et al., 2004;
Beyret and Lin, 2011). piRNA expression is massively induced in pachytene spermatocytes
and this specific subset of piRNAs is generally called the pachytene piRNAs (Aravin et al.,
16
2006; Grivna et al., 2006). Pachytene piRNAs are different from fetal piRNAs or pre-
pachytene piRNAs that bind MILI and are expressed before the pachytene stage of the first
meiotic prophase (Chuma and Nakano, 2013). The production of pachytene piRNAs has
been demonstrated to be independent of the ping-pong amplification mechanism (Fig. 3).
Instead, pachytene piRNAs are more likely to be produced by primary processing pathway
(Beyret et al., 2012).
Deletion of Miwi gene in mouse results in arrest of spermatogenesis at round spermatid
stage with no obvious defects in meiotic progression and complete lack of elongating
spermatids (Deng and Lin, 2002). The expression of pachytene piRNAs is significantly
reduced in the Miwi knockout testis (Grivna et al., 2006) suggesting that their expression is
dependent on MIWI function. Members of the Argonaute family including PIWI proteins are
small RNA-guided nucleases that can cleave target nucleic acids (Ender and Meister, 2010).
Interestingly, a mutant knock-in mouse line expression catalytically inactive form of MIWI
devoid of RNA slicing activity manifest almost identical spermatogenic phenotype than the
Miwi full knockout mouse, which indicates that a substantial portion of the biology of MIWI is
implemented via its small RNA-guided slicer activity. This activity includes the post-
transcriptional cleavage of LINE1 (long interspersed nuclear elements) retrotransposon
transcripts (Reuter et al., 2011). The primary piRNA biogenesis was unaffected in the testes
defective in MIWI slicer activity. Therefore, pachytene piRNA production seems to require
MIWI but not the MIWI slicer activity. The mechanism for pachytene piRNA production
remains unclear, however recent studies have started to reveal critical components, such as
Zucchini/MitoPLD/PLD6 as discussed above, and an RNA helicase MOV10L1 that has been
demonstrated to act upstream of PIWI proteins in the primary processing of pachytene
piRNAs by knockout mouse models (Zheng and Wang, 2012; Zheng et al., 2010; Frost et al.,
2010). Primary piRNA biogenesis has been also suggested to involve 3´ to 5´ exonucleolytic
processing for maturation of piRNA 3´end, after the precursor transcript with processed
5´end has been loaded to PIWI proteins (Vourekas et al., 2012).
The functions of the pachytene piRNAs and the reason of their overwhelmingly high
expression are still under active investigation. As discussed above, piRNAs can guide MIWI-
mediated slicing of TEs (Reuter et al., 2011). However, pachytene piRNA population is
diverse and only around 20% of piRNAs map to TE genes. The majority of pachytene
piRNAs cluster in repeat-devoid intergenic regions and a fraction maps also to protein-coding
genes (RefSeq exons). High-throughput sequencing after cross-linking and
immunoprecipitation (HITS-CLIP) of MILI and MIWI bound RNAs coupled with RNA-
sequencing revealed that the majority of the intergenic and uniquely mapped RefSeq-exon
17
piRNAs had no complementary larger RNA targets (Vourekas et al., 2012). This suggests
that MILI and MIWI proteins do not use piRNAs as guides to target non-repeat RNAs. It is
currently unknown for what purpose these piRNAs without complementary targets are
produced, and one possibility is that they are the end products of a degradation mechanism
acting on e.g. meiotic RNA transcripts that are no longer required in haploid cells (Vourekas
et al., 2012). The functional characterization of pachytene piRNAs is challenging due to the
complexity of spermatogenic system and the lack of in vitro culture model for meiotic and
post-meiotic differentiation. Moreover, proteins that function in piRNA pathways seem to have
other piRNA-independent functions as well. For example, MIWI has been demonstrated to
bind, protect and translationally repress spermiogenic mRNAs without using piRNAs as
guides (Vourekas et al., 2012; Nishibu et al., 2012).
5.3. Epigenetic inheritance and non-coding RNAs
Transgenerational effects have wide-ranging implications for human health, biological
adaptation and evolution, and germ line cells play a key role in this process. In addition to
genetic information, the epigenetic information can be transmitted from parent to offspring via
the gametes, which enables transgenerational epigenetic inheritance. Because the
environmentally induced changes in the epigenome of the germline become permanently
programmed, the altered epigenome and phenotype can be transmitted to subsequent
progeny. Environmental epigenetics has certainly a critical role in disease etiology (Skinner,
2011). DNA methylation has always been considered to be a likely candidate for mediating
transgenerational epigenetic inheritance because of vast changes of DNA methylation
patterns during early mammalian development (Lange and Schneider, 2010). However,
recent evidence points towards diffusible factors, in particular RNA, as a possible mechanism
(Daxinger and Whitelaw, 2012). Non-coding RNAs have the ability to modify epigenetic
marks, and therefore could mediate epigenetic inheritance through homology-dependent
regulation. Mature sperm contain different RNA populations, and sperm-borne RNA has been
detected in the zygote, and it is easy to envisage that RNAs transmitted from sperm to early
embryo at fertilization, could act as mobile signals that influence the epigenetic status of
certain elements (Daxinger and Whitelaw, 2012).
piRNAs that have a role in the silencing of mobile genetic elements are also considered as
potential mediators of epigenetic transgenerational inheritance (Aravin et al., 2008;
Kuramochi-Miyagawa et al., 2008; Daxinger and Whitelaw, 2012). Recent reports show that
in C. elegans germ line, piRNAs can initiate a multigenerational epigenetic memory of RNA
that is recognizes as “nonself” (Shirayama et al., 2012; Ashe et al., 2012; Lee et al., 2012).
18
By using the RNA-based recognition mechanism, foreign sequences could be detected not
by any molecular signature, but by comparing the foreign sequence to a memory of previous
gene expression. On the other hand, endogenous germline-expressed genes are actively
protected from piRNA-induced silencing (Lee et al., 2012). This raises an intriguing possibility
that mammalian small RNAs could function in epigenetic programming and generation of an
epigenetic memory.
6. Future perspectives
The ability of germ cells to transmit their genetic and epigenetic information to following
generations highlights the importance of the correct establishment and maintenance of
epigenetic marks, as well as the protection of information to ensure the quality and to prevent
the transmission of faulty information to offspring. Extensive and accurate transcriptional and
post-transcriptional regulation is required to support the highly orchestrated expression of
specific genes required for each step of spermatogenesis. In addition, spermatogenesis is
accompanied by major chromosomal rearrangement during meiosis and haploid cell
differentiation. The combined actions of miRNAs, endo-siRNAs and piRNAs, and possibly
several other classes of yet unidentified small RNAs have major contribution to the control of
gene expression and chromatin in male germ cells. The functions of small RNAs, especially
those of piRNAs are diverse and require still extensive long-term investigation before we are
able to obtain the full picture. In the future it is also essential to identify the regulatory
network governing the expression of small RNAs, as well as to clarify the importance of the
spatial control of RNA regulation in specific subcellular compartments.
People in Europe and other developed countries have been lately challenged by increasing
problems in fertility. Both genetic factors and environmental interference contribute to the
etiology of subfertility and infertility. Male infertility is often reflecting faults in
spermatogenesis, and these faults are usually overcome by assisted reproductive
technologies. We know that the epigenetic reprogramming taking place during the whole
course of spermatogenesis is important for correct development of spermatozoa. However,
we do not understand the possible far-reaching transgenerational consequences resulted
from the bypassing of the critical differentiation steps. Since small RNAs act as versatile
regulators of gene expression and epigenetic events during spermatogenesis, they may
provide a potent tool for the diagnosis of infertility and the assessment of the suitable
methods for infertility treatments.
19
Acknowledgements We want to thank all the lab members for their comments and critical reading of the
manuscript. The authors are funded by the Academy of Finland, Emil Aaltonen Foundation,
Novo Nordisk Foundation and Sigrid Jusélius Foundation.
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Figure captions:
Fig. 1. Male germ cell differentiation. Spermatogenesis is initiated after birth by mitotic
proliferation of spermatogonia that finally enter into the meiotic program and become
spermatocytes. After long meiotic prophase I that includes synaptonemal complex formation,
crossing over, and homologous recombination, spermatocytes undergo two fast divisions (MI
and MII) to produce haploid round spermatids (RS). Post-meiotic differentiation is
characterized by dramatic morphological changes including chromatin compaction by
histone-protamine transition, nuclear reshaping, and acrosome and flagellum formation. The
transcriptional activity is high until elongating spermatids (ES). After histone-protamine
transition, transcription in late ESs is largely silenced and protein production in these cells is
mostly dependent on mRNAs that are transcribed earlier and translationally regulated in RNP
complexes. The timing of Cre-mediated gene deletion in different germ cell-specific Dicer1
and Drosha knockout mouse models discussed in this review are shown by arrows. The
expressions of fetal, pre-pachytene and pachytene piRNAs are also indicated.
Fig. 2. Biosynthesis and functions of Dicer-dependent small RNAs. The nuclear
microprocessor complex that comprises endonuclease Drosha and DGCR8 processes the
nascent hairpin loop primary miRNA (pri-miRNA) transcripts into 70-nucleotide pre-miRNAs
that are transported to the cytoplasm. The pre-miRNAs are further processed by the
endoribonuclease Dicer into 21-25 nucleotide small dsRNAs that subsequently unwind and
associate with RNA-induced silencing complex (RISC). Endo-siRNAs are processed by long
double-stranded RNA precursors and their processing does not require Drosha activity. Small
RNAs mediate sequence-specific binding of RISC complex to their target mRNAs that are
directed either to cleavage or translational regulation. Small RNAs are also known to have
nuclear effects on gene expression at transcriptional level.
Fig. 3. piRNA pathways in murine male germ cells. piRNA precursors are transcribed from
piRNA clusters and transported to the cytoplasm for further processing in the cytoplasmic
germ granules. In fetal male germ cells, primary piRNAs produced by primary processing
pathway are loaded to MILI. The response is amplified by the secondary processing pathway
(ping-pong cycle) including cyclic production of MILI-bound primary piRNAs and MIWI2-
bound secondary piRNAs. This pathway is exploited in fetal prospermatogonia to silence
transposon expression. MIWI2 can also be imported to the nucleus where it controls the
methylation of transposon genes. In meiotic and post-meiotic cells, piRNAs are produced
29
only by primary processing pathway and are loaded to MILI and MIWI. The targets and
mechanisms of these post-natal piRNAs are still under investigation.
30
Birth
Stra8Cre
Ddx4Cre Ngn3Cre TnapCre
TRANSCRIPTIONAL ACTIVITY
HISTONES
pachytene piRNAs
Prm1Cre
ACTIVE TRANSLATIONAL REGULATIONfetal
piRNAs pre-pachytene piRNAs
PGCs Prospermatogonia Spermatogonia Spermatocyte M-I and M-II RS ES Spermatozoa
PROTAMINES
31
Pri-miRNA Pre-miRNA
NUCLEUS
CYTOPLASM
DGCR8 Drosha
endo-siRNAs
Dicer
target mRNA
RISC AGO
RISC
AGO
Translational repressionmRNA cleavage
Pre-miRNA
Transcription
?
Small RNA gene
21-25 nt small RNA
32
piRNA loci
NUCLEUS
CYTOPLASM
Primary processing
MIWI2
MOUSE GERM CELL
Transcription
Sense transcripts
MILI
MILI
MILI MIWI2
MIWI
Post-natal
germ cells
Fetal prospermatogonia
Antisense transcripts
Primary piRNAs Secondary piRNAs
Secondary processing
ping-pong cycle
33
Table 1. Male germ line-specific Dicer1 and Drosha knockout mouse models.
Mouse model Cre expression Spermatogenic phenotype References
TnapCre-Dicer1
Primordial germ cells (PGCs)
Defects in PGC proliferation and post-natal spermatogenesis.
(Hayashi et al., 2008; Maatouk et al., 2008)
Ddx4Cre-Dicer1
Prospermatogonia (E18)
Meiotic defects, reduced number of haploid cells, drastic abnormalities in head and tail morphology of the remaining spermatozoa.
(Liu et al., 2012a; Romero et al., 2011)
Ngn3Cre-Dicer1 Spermatogonia
No gross abnormalities in spermatogonia and spermatocytes. Major defects in haploid differentiation including chromatin organization, condensation and nuclear shaping of elongating spermatids.
(Korhonen et al., 2011)
Stra8Cre-Dicer1 Spermatogonia
Morphological disruptions of spermatocytes and round spermatids, impaired meiotic progression.
(Wu et al., 2012; Greenlee et al., 2012)
Stra8Cre-Drosha Spermatogonia
Severe morphological disruptions in spermatocytes and round spermatids. Stronger phenotype than for the Stra8Cre-driven deletion of Dicer1 gene.
(Wu et al., 2012)
Prm1Cre-Dicer1 Spermatids
Defects mainly in post-meiotic differentiation, abnormal head morphology.
(Chang et al., 2012)
34
Small RNAs in spermatogenesis Ram Prakash Yadav and Noora Kotaja
Highlights
Post-transcriptional gene regulation has important role in spermatogenesis
Small RNAs are essential regulators of gene expression
Small RNAs function at post-transcriptional or epigenetic level
Male germ cells express many classes of small RNAs (miRNAs, endo-siRNAs,
piRNAs)
Knockout mouse models have revealed the importance of small RNAs in
spermatogenesis