Retracted article: In vitro derivation of mammalian germ cells from stem cells and their potential therapeutic application.

Pluripotent stem cells (PSCs) are a unique type of cells because they exhibit the characteristics of self-renewal and pluripotency. PSCs may be induced to differentiate into any cell type, even male and female germ cells, suggesting their potential as novel cell-based therapeutic treatment for infertility problems. Spermatogenesis is an intricate biological process that starts from self-renewal of spermatogonial stem cells (SSCs) and leads to differentiated haploid spermatozoa. Errors at any stage in spermatogenesis may result in male infertility. During the past decade, much progress has been made in the derivation of male germ cells from various types of progenitor stem cells. Currently, there are two main approaches for the derivation of functional germ cells from PSCs, either the induction of in vitro differentiation to produce haploid cell products, or combination of in vitro differentiation and in vivo transplantation. The production of mature and fertile spermatozoa from stem cells might provide an unlimited source of autologous gametes for treatment of male infertility. Here, we discuss the current state of the art regarding the differentiation potential of SSCs, embryonic stem cells, and induced pluripotent stem cells to produce functional male germ cells. We also discuss the possible use of livestock-derived PSCs as a novel option for animal reproduction and infertility treatment.

Introduction

Approximately 50–60 % of human infertility is caused by defects in the male germ line [1]. Current infertility treatments include intrauterine insemination, ovulation induction for in vitro fertilization, and intracytoplasmic sperm injection (ICSI), which usually is associated with low efficiency and unwanted side effects in the offspring most likely caused by epigenetic aberrations [2]. However, these treatments are available only to patients who are able to produce functional gametes.

Stem cells are pluripotent cells that have the capacity for indefinite self-renewal and can generate multiple cell types with specific functions in the body [3]. Spermatogenesis is an intricate process that starts with self-renewal of spermatogonial stem cells (SSCs) and leads to fully differentiated functional haploid spermatozoa (Fig. 1). Perturbations at any stage of spermatogenesis may result in infertility; because the process is error prone, and defective sperm production is thought to be responsible for 15–50 % of all infertility cases [2].

Fig. 1

Schematic representation of differentiation of mammalian PSCs into germ cells in vitro. The totipotent zygote is the earliest cell. The ICM in blastocysts contains all cell types forming the entire organism, and ESCs have been established from ICM cells under suitable in vitro culture conditions. Following germ line specification, PGCs appear first in the extraembryonic mesoderm. The germ line potential is preserved during embryo development in OCT4+ cells located in ICM cells of the blastocyst, epiblast stem cells, PGCs, and gonocytes in male gonads. Both ESCs and iPSCs can be differentiated to PGC-like cells under in vitro culture conditions with BMP4 and/or GDNF. The development of germ cells, already during the postnatal period, is sex-specific. Male germ cells enter mitotic arrest and are reactivated to initiate spermatogenesis after birth. Female germ cells enter meiosis and undergo meiotic arrest until after birth [80, 83]. eBlastocyst equine blastocyst, mESCs mouse embryonic stem cells, mEpiblast mouse epiblast, hiPSCs human induced pluripotent stem cells

Oct4 expression is critically involved in the regulation of pluripotency and is found in the inner cell mass (ICM) of blastocysts, the epiblast, and the primordial germ cells (PGCs), but is repressed in somatic cells [4]. PGCs migrate through the hindgut to the genital ridge, where the ovaries and testis are formed. After termination of migration, PGCs start to express a marker gene for post-migratory germ cells, Ddx4 (mouse vasa homologue: Mvh) [5], which initiates sex-specific development. Following migration, male PGCs enter mitotic arrest, and after birth, male germ cells are reactivated to start spermatogenesis. By day E15.5, oogonia are formed in females and gonocytes are formed in males. Gonocytes persist until shortly after birth, and SSCs are formed between postpartum days 0 and 6 in male mice. The transition of gonocytes to SSCs lasts several months in livestock and years in humans and other primates [6].

Male germ cells grown from gonocytes continue to self-renew as SSCs throughout life. SSCs from neonatal and adult mice can develop into pluripotent stem cells (PSCs) when cultured under specific conditions in vitro [7, 8]. The establishment of human adult germ line stem cells from human testicular tissue has been reported [9, 10].

Here, we review the current status of the differentiation potential of SSCs, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) towards male germ cells. We discuss their potential for use in reproductive medicine and for gaining a better understanding of stem cell development and spermatogenesis. In addition, we discuss the potential use of large domestic animal-derived PSCs for drug screening, infertility treatment, production of genetically modified (GM) livestock, and human disease models.

Male germ cell generation in vitro

In the past decade, significant progress has been made in the derivation of male germ cells from various types of stem cells. Currently, two approaches are used for generating male germ cells from PSCs: (1) in vitro differentiation to haploid cells, and (2) a combined approach by using in vitro differentiation and in vivo transplantation.

Two main sources of PSCs exist in early mammalian embryos: the ICM of preimplantation blastocysts and the epiblast of pre- and post-implantation embryos, which are termed ESCs and epiblast stem cells (EpiSCs), respectively [11-13]. Mouse embryonic stem cells (mESCs) can be differentiated into all types of cells, including PGCs and undergo further differentiation and meiosis to immature gametes, which in turn form blastocysts after fertilization [14, 15]. Several groups have reported the delivery of live pups from in vivo differentiated sperm cells [16, 17]. A similar developmental capacity was proposed for human and primate ESCs [18-22]. HESCs and hiPSCs are capable of differentiating into the three germ layers and into germ cells. Human iPSCs have been used as a model system to understand the genetic and epigenetic basis of germ cell specifications [23], and germ cell-like cells could be derived by in vitro induction.

It is known that hESCs are more similar to mouse EpiSCs than mESCs [13]. Two different pluripotency states are represented by these cell types: (1) a naïve state, which is characteristic of mESCs, and (2) a primed pluripotent state, which is typical for EpiSCs and hESCs. These cells do not have the capacity to form germ cell line-competent chimeras upon injection into blastocysts [24]. In the laboratory mouse, a properly primed pluripotency state is associated with the induction of an epiblast-like state prior to germ cell derivation, whereas in humans, the correct entry into meiosis led by RNA-binding proteins seems to be the major obstacle (Fig. 2).

Fig. 2

Schematic model of germ cell derivation in vitro. a Mouse embryonic stem cells (mESCs) or mouse induced pluripotent stem cells (miPSCs), in general PSCs, can be induced into an epiblastic-like (mEpi-like) cells which are able to respond to the signaling pathway started by BMP4 [17, 120, 121]. A primordial germ cell (PGC)-like cells are induced and these cells, in an appropriate in vivo microenvironment (i.e., transplantation into neonatal mouse testis or ovarian bursa) become functional spermatocytes or oocytes. After intracytoplasmic sperm injection (ICSI) these gametes generate fertile and healthy offspring of both sexes. b Human pluripotent stem cells (hPSCs) either human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) present a primed pluripotency state, more similar to a mEpi-like cells, and they can directly respond to BMP4 signaling to attain a PGC-like status [122–126]. PGC-like cells need the presence of different RNA-binding proteins, to progress meiosis and form haploid cells in vitro after induction by retinoic acid (RA) and to express the correct spermatogonial markers when subjected to in vivo microenvironment control after xenotransplantation in immunosuppressed mouse testes. SSC spermatogonial stem cell, Spg spermatogonia

IPSCs have been generated by over-expression of various combinations of transcription factors (e.g., OCT4, MYC, KLF4, and SOX2) in a broad range of species [25, 26]. Recent reports have shown that hiPSCs can enter meiosis and, in some cases, produce haploid products [27-30]. By contrast, the differentiation potential of ESCs and iPSCs to germ cells has not been reported in livestock animals.

Recently, endocrine disruptors have been suggested to have profound trans-generational effects on male germ cell function and have been associated with infertility and tumor formation [31-35]. Exploitation of in vitro culture systems to support mammalian germ cells might improve the development of novel methods for monitoring putative detrimental effects of reproductive toxicants. We have demonstrated that bovine testicular iPSCs are useful for screening the toxicity of environmental disruptors, such as phthalate esters by examining their effects on the maintenance of stemness and pluripotency, and for identifying signaling pathways that might be affected by disruptors [36, 37]. Modeling spermatogenesis in vitro has been employed to examine the effects of environmental toxicants on the differentiation process to spermatozoa [38]. This represents a unique platform for assessing the toxicity of various environmental disruptors on human reproductive functions in a rather straightforward manner.

Restoring fertility following SSC transplantation into the testis

The most direct assay to confirm the biological capacity of SSCs is functional transplantation. Re-transplantation of SSCs obtained from testicular biopsies restored fertility in infertile recipient mice [39-45]. For SSC transplantation, a donor testis-derived cell suspension is injected into the seminiferous tubules of a recipient male, in which the endogenous germ cells have been depleted by treatment with chemotoxic drugs (e.g., busulfan), or it is injected into an animal that is naturally devoid of germ cells (e.g., W/Wv mutant males). Successful transplantation of SSCs with the production of viable spermatozoa has also been reported in livestock animals, including pigs, cattle, sheep, and goats [46-49]. Functional sperm derived from sheep and goat SSCs in the host testis produced donor-derived progeny [48, 54]. SSC transplantation is the only method for identifying fully functional SSCs and confirming their biological activity.

The testicles are an immune-privileged site that is crucial for successful allogenic SSC transplantation between unrelated, immunocompetent individuals [46, 48, 50]. In nonhuman primates, treatment with a humanized monoclonal antibody against CD154 prevented acute renal allograft rejection [51]. SSC transplantation leads to restoration of fertility in males after successful tumor treatment, suggesting SSC transplantation as an emerging clinical application [43, 52–56]. Recently, SSCs were successfully transplanted into the testes of recipient macaques that had been treated with busulfan to destroy the endogenous sperm cell population [57]. The donor genotypes were found in ejaculated sperm of the recipients and mature ejaculated sperm led to blastocyst development after ICSI in Rhesus oocytes, clearly indicating functional spermatogenesis in the foster testes that had been rendered sterile by prior chemotherapy. Thus, in cases of a deficient testicular environment or in the absence of differentiated haploid germ cells or spermatozoa, SSC transplantation may be a valuable therapeutic option to restore fertility. The findings in large animals and nonhuman primates are promising for the application of transplantation of human SSCs; for example, tissue biopsies obtained from adolescent male patients prior to chemotherapy may be stored to produce functional germ cells for later use after successful cancer treatment [57, 58].

Enhancement of SSC self-renewal and stemness in vitro

The core ESC regulatory transcription factors that regulate self-renewal and pluripotency include OCT4, SOX2, and NANOG [59-64]. Expression of Oct4, Sox2, Klf4, and c-Myc, rather than Nanog, was observed in mouse SSC in vitro, but tumor formation after transplantation was not observed [65]. NANOG expression was shown to be essential for PGC maturation in the genital ridge during fetal development [66]. In our studies, bovine testicular cells did not express endogenous OCT4, NANOG, or SOX2; instead, they expressed KLF4 and c-MYC [67]. By contrast, bovine iPSCs expressing pluripotency markers, including OCT4, NANOG, SOX2, STAT3, c-MYC, KLF4, TERT, and DNA methyltransferase 3 (DNMT3) have been reported; benign cystic teratomas containing derivatives of the three germ layers were observed after subcutaneous transplantation into nude mice [36, 37]. These data suggest that NANOG plays a critical role in the ability to contribute to teratoma formation as an ultimate proof of pluripotency. Speculatively, silencing of NANOG expression may be essential for maturation of SSCs from PGCs or gonocytes.

Sato et al. [65] demonstrated the derivation of functional sperms from mouse SSCs using an in vitro organ culture system. The cells were cultured in explanted neonatal testis tissues, and sperm cells could be differentiated from SSCs; ultimately viable sperm gave rise to offspring after micro insemination. These results seem to be applicable to other species, including humans and large domestic species. The technology requires explant culture with testicular tissue to serve as host incubator [66], which, however, may pose additional challenges related to hygiene and variability. In contrast, human SSCs that were cultured in medium supplemented with retinoic acid and stem cell factor can differentiate into haploid spermatids that were microinjected into mouse oocytes and showed evidence of fertilization potential [67].

Progress in stem cell technologies might lead to new cell-based infertility treatments if immunologically compatible patient-specific cells can be derived. Using SSCs for autologous cell-based therapy would be superior to ESC-based treatments, because it avoids the ethical problems associated with the use of human ESCs. Moreover, studies on SSCs may offer unique insight into one of the earliest fate decisions of ESCs or EpiSCs and into the biology of SSCs, which are of fundamental importance for the continuity of species [6].

PSCs to screen for environmental toxicant-associated male infertility

Numerous studies have confirmed that environmental endocrine disruptors have adverse effects on male fertility; phthalate derivatives lead to testicular atrophy, decreased testicular weight and lower testosterone level [68-71]. The detachment of germ cells from the seminiferous epithelium and the increased incidence of germ cell apoptosis have been observed in young peripubertal rodents after exposure to mono-(2-ethylhexyl) phthalate (MEHP) [71]. The number of germ cells was significantly reduced in cultured human fetal testes after exposure to 10−4 M MEHP for 3 days, mainly associated with a dramatic increase in apoptosis [72]. The toxicity of environmental disruptors such as cadmium [73], MEHP [74], and uranium [75], was investigated using organ culture systems with human fetal testes. Thus, the use of hESCs and iPSCs is promising for monitoring potentially detrimental effects of environmental disruptors.

Bovine iPSCs and testicular cells have been successfully used as in vitro models to study the toxicity of phthalate esters. We found that bovine iPSCs were more resistant to androgen receptor (AR)-dependent apoptosis than testicular cells, most likely attributed to regulation of the AR-p21Cip1 cascade via p53, which showed significantly enhanced expression. Phthalate esters significantly reduced AR expression in bovine iPSCs. Collectively, these studies indicate that iPSCs may be useful for screening for adverse effects from endocrine disruptor [36, 37]. This screening system has also promised as a useful model for studying the effects of environmental factors on human germ cell development.

Derivation of gametes from mammalian adult tissues, and germ line cell differentiation from ESCs and iPSCs

Functional adult germ line stem cells can be derived from human testes and adult mouse and human ovaries [9, 10, 76–78]. However, stem cells from human testicular tissue did not form teratomas after transplantation into immune-deficient mice, suggesting limited pluripotency [9, 10]. Mitotically active oogonial stem cells could be isolated from the surface of mouse adult ovaries and human ovarian tissues by sorting for DDX-expressing cells [78]. However, other investigators did not find mitotically active female germ line progenitors in mouse ovaries. Moreover, Ddx4-expressing cells from postnatal mouse ovaries did not enter meiosis and did not develop to oocytes during de novo folliculogenesis under their experimental conditions [79]. Gamete derivation in vitro from PSCs is challenging because many PGC markers are identical to PSC markers [80], which makes it extremely difficult to discriminate early embryonic germ line cells from PGCs.

Hübner et al. [81] were the first to report the in vitro gamete production from mouse ESCs carrying the Oct4 reporter gene. Ovarian follicle-like structures were observed under culture conditions without feeder layer or growth factors. Toyooka et al. [14] described for the first time the derivation of male germ cells from mouse ESCs carrying a Ddx4 (Mvh) reporter construct. These authors used embryoid bodies (EBs) as the starting material and induced EBs to differentiate in suspension culture in the absence of leukemia inhibitory factor (LIF). Ddx4+ cells gradually appeared in the EBs, suggesting the presence of cells with the characteristics of post-migratory PGCs in EBs. Subsequently, purified Ddx4+ cells were transplanted together with male genital ridge cells into adult mouse testes. The cell aggregates formed seminiferous tubules that supported complete spermatogenesis derived from purified Ddx4+ cells. This study clearly demonstrates that germ line specification and the emergence of post-migratory PGCs occur spontaneously or are induced in EBs. However, spermatozoa derived from PGCs could not activate oocytes. Male PGCs could be derived from mouse ESCs in vitro with the aid of EBs [15]. The cells spontaneously became post-meiotic and were capable of activating oocytes after injection of PGC-derived male haploid cells into EBs, using an antibody that specifically reacted with specific stages of postnatal male germ cells up to spermatozoa [15].

Nayernia et al. [16] reported the induction of male gametes from ESCs and the successful production of offspring derived thereof. However, the low viability and growth abnormalities in the progeny derived from in vitro-derived germ cells indicated imprinting errors, suggesting erroneous epigenetic reprogramming associated with the development of male-specific germ cells under in vitro conditions. Moreover, the remaining undifferentiated stem cells in culture might cause teratomas after transplantation. Further investigations into the epigenetic reprogramming status in induced germ cells might provide valuable information regarding sex-specific germ cell differentiation in vitro. In vitro germ cell induction mechanisms have not yet been sufficiently defined to allow for examining the normal development of germ cells ex vivo. Further in vivo studies are needed to establish the effectiveness of in vitro systems as a reliable assay of germ cell development [80].

The expression profiles of marker genes in germ cells and PSCs may provide important information for deriving germ cells from those cells. Marker molecules for specific types of stem cells are shown in Table 1. Basic fibroblast growth factor (bFGF) and feeder cells increased the expression of PGC marker genes such as VASA (DDX4), DAZL, and OCT4 in human germ-like cells differentiated from hESCs [85]. Tilgner et al. [20] reported the enrichment of putative PGCs from hESCs that had been sorted using an antibody specific for stage specific embryonic antigen-1 (SSEA-1). Gelatin-bound monolayers are obviously a robust system for generating large number of differentiated cells. However, these cells do not enter meiosis.

Table 1

Gene and surface marker expression profiles of pluripotent stem cells and germ cells

ND not determined or no information, m mouse, h human, b bovine, ESCs embryonic stem cells, iPSCs induced pluripotent stem cells, PGCs primordial germ cells, SSCs spermatogonial stem cells, AP alkaline phosphatase

aPluripotency markers

bGerm cell markers

Transplantation of ESC-derived somatic cells or tissues is promising for curing many human diseases. However, derivation of gametes from unrelated ESCs is associated with incompatibilities of the immune systems. Well-characterized iPSCs may be a good option for obtaining sufficient numbers of autologous cells. HiPSCs could be successfully differentiated to post-meiotic cells without over-expression of germ line-related transcription factors [26]. Cells were cultured without bFGF as monolayers for 3 weeks and the pluripotency markers SSEA-4 and OCT4 were down-regulated at the end of this period. Under these conditions, male germ-like haploid cells were obtained from hiPSCs. Tilgner et al. [20] demonstrated for the first time the meiotic competence of hiPSC-derived cells, which suggests the possibility of producing human gametes in vitro. The ability of hiPSCs and hESCs to differentiate into presumptive SSC-like cells in vitro, and to contribute to advanced spermatogenesis, including round spermatids, was reported recently [30]. However, round spermatids could not fertilize human oocytes. The feasibility and safety of the culture systems will need to be established in animal models.

Mouse ESCs and iPSCs can be induced to form epiblast-like cells that, in turn, develop into PGC-like cells when the culture medium is supplemented with BMP4 [17] (Table 2). The resulting PGC-like cells were then transferred to the testes of infertile mice and produced sperm that were used for ICSI; transfer of the resulting embryos into recipient females gave rise to viable offspring. This is the most advanced protocol for the deviation of functional gametes from PSCs until now. Further experiments are required before this system could be used for therapeutic treatments in human patients because some of the offspring showed malignant tumors in the neck area [17]. Human iPSC-derived cells should be monitored carefully to eliminate mutations, specifically in tumor suppressor genes [83, 84].

Table 2

In vitro germ cell-like derivation from pluripotent stem cells

Animals	Type of pluripotent stem cells	Methods	Germ cell-like formation	References
Human	ES cells	Human BMP4, BMP8a, DAZ2, DAZL, BOULE, RA	Germ cell-like cells	[30]
Human	ES cells and iPS cells	Human BMP4, BMP8a, VASA, RA	Germ cell-like cells	[126]
Human	iPS cells	Human BMP4, BMP8a, DAZ2, DAZL, BOULE, RA	Germ cell-like cells	[31]
Human	iPS cells	Human BMP4, BMP8a, VASA, and transplantation into murine seminiferous tubules	Induced PGCs	[127]
Human (deletions in the Y chromosome)	iPS cells	Human BMP4, BMP8a, transplantation into murine seminiferous tubules	Induced PGCs	[128]
Human	Umbilical cord Wharton’s jelly-derived mesenchymal stem cells (HuMSCs)	Human MSCs → bFGF, EGF → 5–7 days co-cultured with sertoli cells (1–3 weeks)	Male germ-like cell	[124]
Human	ES cells	Activin A+BMP4VASA	Germ-like cells	[125]
Mouse	ES cellsiPS cells	Differentiation to EpiLCs with bFGF + ActivinA → BMP4 → PGCLC generation → transplantation into neonatal mouse testis	Sperm-like cells	[19]
Mouse	ES cellsiPS cells	Differentiation to EpiLCs with bFCF + Activin A → BMP4 → PGCLC generation → transplantation into neonatal mouse ovarian bursa	Oocyte-like cells	[122]
Mouse	ES cellsiPS cells	Differentiation to EpiLSCs with bFGF + Activin A → Prdm1, Prdm14, TFAP2C → PGCLC generation	Sperm-like cells	[123]

Epigenetic control of germ cell development

A bimodal pattern of DNA methylation has been detected during the specification and maturation of mouse male germ cells (Fig. 3). PGCs derived from the epiblast at E6.5–E7.5 are stimulated by BMP4, then migrate from the epiblast to the hindgut at E7.5–E9, and finally to the genital ridge at E9.5–E11.5. In E6.5 mouse embryos, PGCs show DNA hypermethylation with repression of certain genes [85]. The epigenetic marks are erased during migration of PGCs [86], particularly in imprinted genes and transposons of PGCs. The re-establishment of DNA methylation in germ cells initiates from the formation of pro-spermatogonia or gonocytes. Although DNA methylation is acquired during the prenatal mitotic arrest of the gonocytes, de novo and maintenance of methylation occur only during mitosis of spermatogonia and meiotic prophase I, whereas maintenance methylation appears only during mitosis [87] (Table 3). The global erasure of DNA methylation also occurs during early embryonic development [88, 89].

Fig. 3

Schematic diagram reveals the expression of DNA methylation profiles in mammalian spermatogenesis. Bimodal DNA methylation patterns in male germ cell development. PGCs are derived from the epiblast at E6.5 and migrate to the genital ridge. During migration, the epigenetic marks are widely erased. After erasure of the DNA methylation marks, reestablishment of the male germ cell DNA patterns initiates from prospermatogonia to entering meiosis. After fertilization, DNA patterns are broadly erased by active demethylation, whereas the imprinted genes are maintained by DNMT1 activity

Table 3

miRNA that plays a regulatory role in spermatocyte meiosis and spermatogenesis

MiRNA	Targets	Expression	Function	References
Rhesus monkey and mouse testis
miR449	MECP2, ASB1, BCL2, NOTCH1, CASP2, FITLG, VCL, FOXJ2, INHBB, BOX11, CCNE2, GMFB and DLL1	Up-regulation in testisLocalized to spermatocytes and spermatids	Represses the proliferation of a germ cell line	[129]
miR34b	NOTCH1, LGR4, VEZT, MAN2A2, FOXJ2	Up-regulation in testis	Regulates the germ cell proliferation and survival	[130, 131, 133]
Mouse testis
miR34a	CCND2, BLC2, GMFB, SIRT1	Up-regulation from day 7 to day 14 in mouse testis	Represses proliferation, promotes apoptosis	[130–132]
miR34c	CCND3, CCNG1, CCNB1, CCNC, CCNE1, CDK4, CDK6, E2F5, Fos, CDC2, TGIF2, NOTCH2, STRBP, LBR4, KFFL, NOTCH1, PPP1LL, GALT, KITLG, SDA94, CCNL1, ZFD148, GMFB	Highly expressed in pachytene spermatocytes and round spermatids	Cycle regulatormGSC apoptosisSSC differentiation	[130, 131, 134, 135]
miR184	NCOR2	Localized in the germ cell of mouse testis	Promotes the proliferation of germ cell line	[136, 137]
miR24	MBD6, H2AX	Pachytene spermatocytes	Meiosis	[136]
miR214	WDTC1, HS proteins	Pachytene spermatocytes	Meiosis	[106, 136]
miR320	Protocadherins	All germ cells	Cell adhesion	[106, 136]
miR469	TP2 and PRM2	Pachytene spermatocytes and round spermatids	Regulates the chromatin remodeling	[106]
miR18	HSF2	Highly expressed in spermatocytes	Male germ cell maturation	[108]
miR122a	TNP2	In late stage male germ cells	Chromatin remodeling	[135]
mir355	Rsbn1	Up-regulation in adult testis	Transcriptional regulation	[130]
miR181b	Rsbn1	Up-regulation in adult testis	Transcriptional regulation	[130]
miR181c	Sox5, Sox6, Rsbn1	Up-regulation in adult testis	Transcriptional regulation	[130]
miR185	RhoA, CDC42	In pachytene spermatocytes	Cell cycle regulator	[136]
miR191	BNC2	In beta pachytene spermatocytesDown-regulated in teratozoospermia	Required for normal sperm morphology	[136]

DNMT3-like (DNMT3L) is involved in the maintenance of DNA methylation in stem cells during the quiescent state or during self-renewal of SSCs, whereas DNMT3a and DNMT3b are not involved in this process. In addition to its role in self-renewal, DNA methylation of SSCs may be required for the transition from SSCs to differentiated spermatogonia. DNMT3a and DNMT3b transcripts remain at the highest level in type A spermatogonia compared with other types of male germ cells [90]. Studies into the roles of DNA methyltransferases in SSC differentiation in mice are useful for gaining a better understanding of the underlying biological principles and for the development of new therapies.

Expression of DNMT1, DNMT3a, and DNMT3b is upregulated in leptotene and zygotene spermatocytes during meiosis and spermatogenesis [91]. DNMT1 is present in non-proliferative round spermatids, whereas DNMT3a and DNMT3b maintain the methylation patterns through the de novo methylation pathways, although the roles of DNMT1 in round spermatids remain to be solved. The role of ten–eleven translocation methylcytosine dioxygenase (TET1) has not been elucidated in spermatogenesis, albeit it plays a significant role as a meiotic initiator in oocytes [92]. It remains to be determined whether the biological function of TET1 in spermatogenesis is similar to that in oocytes [92]. In contrast to mouse, human DNMT1, DNMT3a, and DNMT3b are expressed in pachytene spermatocytes [94]. However, in both mice and humans, DNMT1, DNMT3a and DNMT3b are highly expressed in round spermatids [93, 94]. DNMT1 is present in non-proliferative round spermatids, whereas DNMT3a and DMMT3b are expressed after the establishment of the paternal methylation pattern. Thus, DNMT3a2 and DNMT3b may play a role in the de novo methylation pathways, although the role of DNMT1 in round spermatids remains to be solved.

In addition to DNA methylation and demethylation, global changes in histone modifications, such as a decrease in histone H3K9 dimethylation and an increase in histone H3K27 trimethylation, occur in the PGC genome [95, 96]. Although the significance of the global changes in histone modifications remains unclear, it is likely that the alteration is required for the acquisition of potency in the terminal products. A better knowledge on the epigenetic profile during germ cell development is crucial for understanding the underlying biological mechanisms, and thus for developing suitable culture techniques for germ cells, which, in turn, are major prerequisites for developing new therapies with germ cells.

Micro-RNAs (miRNAs) in meiotic and post-meiotic cells

A conditional knockout of Dicer 1 in mice disrupts meiotic and post-meiotic development by decreasing the number of mouse SSCs and by blocking differentiation [97, 98]. In addition, loss of Dicer1 resulted in male infertility in mice [99]. Sertoli cell-specific deletion of Dicer severely impairs sperm competence and leads to male infertility due to the absence of mature spermatozoa and testicular degeneration [97]. Germ cell-specific deletion of Dicer 1 leads to overexpression of genes for meiotic sex chromosome inactivation, to increased spermatocyte apoptosis, and to defects in chromatin organization, the elongation and nuclear shaping of spermatids [100]. These effects suggest that Dicers are crucial for the meiotic and haploid phases of spermatogenesis (Table 3).

MiR-34c expression is up-regulated in spermatocytes and round spermatids trigger apoptosis [101]. This process is at least partially mediated by targeting transcription factor ATF-1 [102]. Thus, miR34c is critical for germ cell development. MiR-469 has been shown to target transition protein 2 (TP2) and protamine mRNAs to be repressed in pachytene spermatocytes and round spermatids [103]. MiR-122a also controls the degradation of TP2 mRNA cleavage [104], and miR-18 can directly target heat shock factor 2 mRNA at the spermatogenesis stage [105].

Collectively, miRNAs play essential roles by regulating each step of male germ cell development, including mitosis, meiosis, and spermatogenesis in rodents. Nevertheless, it remains to be defined which miRNAs are required for the three major stages of spermatogenesis in humans, including spermatogonia, pachytene spermatocytes, and round spermatids [106]. A better understanding these processes may provide new targets for the treatment of male infertility.

In vitro gametogenesis from bovine iPSCs and production of genetically modified (GM) cattle from transgenic iPSCs

Bovine iPSCs established in our laboratory exhibited characteristics similar to those of mESCs with regard to gene expression, transcription factor dependency, and active signaling molecules [36, 37]. Expression of pluripotency markers, including OCT4, NANOG, SOX2, STAT3, c-MYC, KLF4, TERT, and DNMT3A, is maintained in bovine iPSCs (Table 3). Mouse ESCs and iPSCs expressed SSEA-1, but not SSEA-4, whereas human ESCs and iPSCs expressed SSEA-4, but not SSEA-1 (Table 3). Morphology and expression of the SSEA antigens in bovine iPSCs resembled those of mouse ESCs and iPSCs rather than those of human ESCs and iPSCs. Bovine iPSCs express both SSEA-1 and SSEA-4. SSEA-1 expression has been observed in both bovine and equine embryonic stem-like cells [107-109]. The conditions reported by Hayashi et al. [19] may be useful for purifying PGC-like cells from bovine iPSCs (Fig. 1). The availability of functional in vitro culture system is promising for improving breeding of farm animals. The selection process for stud sires aiming to obtaining genetically improved progeny in animal breeding is very expensive and time-consuming. The use of fertile sperm cells derived from iPSCs established from the tissues of neonatal bull calves may be a promising economical option. In addition, stem cell therapies may be useful for restoring fertility in elite bull sires that are unable to produce semen because of physical damage or disease of the testicular somatic environment.

Several attempts have been made to establish germ line-competent bovine ESCs or iPSCs [108-111]; however, so far teratoma formation with derivatives of the three germ layers has not been observed, although it has been confirmed for goat ESCs [112]. Recently, we demonstrated that gene expression could be silenced in bovine iPSCs by using small interfering RNA against p21Cip1, which resulted in the reduced expression of the target genes [36], suggesting the possibility of gene targeting with bovine iPSCs.

Spermatozoa may be useful as vectors for producing GM animals [113-116]. It could be a valuable option in the cattle industry to use spermatids differentiated from genetically modified iPSCs to produce transgenic animals by transplantation into the testes of recipient bull calves or by injecting them into bovine oocytes. We propose to produce transgenic animals by using sperm-like cells differentiated from transgenic iPSCs via in vitro fertilization or ICSI. Bovine SSCs could successfully be propagated in the presence of LIF, epidermal growth factor or fibroblast growth factor 2; however, no full spermatogenesis was established from SSCs transplanted into recipient mouse testis [117]. Complete spermatogenesis has been obtained from autologous transfer of bovine SSCs [47, 48, 118]. Thus, the methodologies described above need significant improvements, and cell-based approaches in livestock reproduction are a challenging task. The derivation of PSCs in livestock is promising for the development of novel disease-resistance strategy, cell or organ therapies, drug screening, and human disease models. It is also important for increasing the efficiency of the livestock industry. For example, dairy manufacturers could derive protein-rich milk from GM cows and thereby reduce the cost of cheese production.

The rapidly emerging DNA nucleases such as ZFNs, TALEN, and CRISPR/Cas may provide additional new options for producing livestock species with targeted genetic modifications with novel traits useful for application in agriculture and biomedicine [119]. There is no doubt that the application of genetic modifications and PSC techniques will improve our understanding of the dynamics of gametogenesis and reproductive biology in general, and will play an important role in the development of novel therapeutic treatments in humans and other mammalian species.

Conclusions

Over the past decade, revolutionary progress has been made in the derivation and characterization of germ cells from various types of stem cells. SSC transplantation in non-human primates is now compatible with functional spermatogenesis in infertile testes after chemotherapy, clearly showing the possibility of using human SSCs from tissue biopsies of adolescent male patients to obtain functional germ cells prior to treatment with high-dose chemotherapy. However, transplantation of human ESC-derived gametes may be associated with incompatibilities of the immune systems, although the testicles constitute an immune-privileged site. Therefore, iPSCs may be a suitable option for supplying sufficient numbers of autologous cells. Differentiated spermatid-like cells from human iPSCs have been unable to fertilize human oocytes until now. More feasible and safer systems must be established in animal models, including large domestic livestock species, to improve the low efficiency of current differentiation protocols and cell viability. From both the academic and therapeutic point of view, in vitro differentiation models using PSCs are highly promising areas. The self-renewal capacity and the pluripotency of stem cells may be valuable in preserving individual genomes and modifying germ lines.

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