**2. Understanding the natural process of spermatogenesis**

#### **2.1 Models to describe spermatogenesis**

medical reproductive assistance [1]. Among half of subfertile couples, male factor is the causative or contributory factor and according to our experience around 12% of subfertile men are severe oligozoospermics or azoospermics. In majority of azoospermia cases, especially in non-obstructive azoospermics, the etiologies are genetically manipulated, and treatment with foci of sperm using assisted reproductive technologies (ART) may carry a greater chance of genetic diseases in the outcome. *In vitro* matured sperm are a greater hope for those men to

Spermatozoa are not only act as a vehicle for delivering paternal DNA to the oocyte, but also robustly contribute to epigenetic processes in embryogenesis. Sperm DNA and the chromatin structure as a unit, drive genes toward activation or silencing upon delivery to the egg [1]. For better performance, it has to undergo several rounds of morphological, biochemical, physiological, and epigenetic changes during spermatogenesis. Even after leaving the testis, sperm is subjected to further maturation in the epididymis, and is not fully competent to do the deterministic task until mixing with accessory gland secretions and activating several proteins, cytokines and signaling pathways. All those processes are interrelated with the stability

Modeling spermatogenesis will ease the study of complex biological interactions *in vitro*, in relation to gametogenesis and fertilization (i.e., epigenetic processes, transcription, translation variations, activation or silence of signaling pathways, etc.). In addition, the modeling will helps to elaborate genetic disorders or other pathological conditions and to discover new drugs (fertility drugs or contraceptive drugs). Rapid development of the field of bioinformatics is immensely useful toward this progress. However, the use of artificial gametes for fertility treatment is far from the vicinity considering the questions still

One of the technical challenges in the study of spermatogenesis is lack of a proper *in vitro* model to recapitulate the process [2]. Recent development of techniques and technologies such as different cell culture systems gene cloning/transgenic animals, gene expression, gene silencing, mass spectrometry and microarray, etc., has immensely been contributed to identify a plethora of endogenous and exogenous factors in the regulation of this process. Compiling all these data in a proper order, for example, grouping expressed genes, proteins, and metabolites into functional categories may allow in recapitulating spermatogenesis process and better understanding of the underlying mechanisms of normal and abnormal pathways [3].

Attempts to make gametes outside the body (*in vitro*) or outside its niche (*ex vivo*) have been ongoing for more than a century [4], and there is a rapid escalation of research in the past decade. Main approaches in achieving this goal include (a) autologous or allogenic testicular transplantation of SSCs, stem cells or differentiated putative germ cells from other sources; (b) auto or xeno-grafting of testicular tissues, SSCs, differentiated putative germ cells into other parts of the body, for example, under the skin; (c) *in vitro* culture of SSCs, stem cells or differentiated putative germ cells with testicular tissues/cells (organ culture systems) or without testicular tissues; (d) sperm cloning, etc. Two main barriers encountered in the *in vitro* spermatogenesis process are; haploidization of stem cells or progression beyond pachytene stage, and inability to further differentiation of the few round spermatids obtained by culture,

attain the biological fatherhood.

26 Spermatozoa - Facts and Perspectives

of DNA it carries.

to be answered.

especially up to formation of tail.

Spermatogonial stem cells represent a very rare population of germ cells consisting about 0.03% (20,000–35,000) in adult mouse testes [5] or even lesser 2000–3000 [6]. Self-renewal of SSCs and spermatogenesis are described using different models. Among them, more detailed studies have been done with mouse. The "A-single" (As ) model originally proposed by Huckins et al., and according to them two types of SSCs are present in the seminiferous tubules; Type A and Type B. Type A—SSCs are more primitive due to absence of heterochromatin, while Type B cells are more differentiated as their nuclear heterochromatin content is high. Type A spermatogonia are subdivided into three groups according to their topological arrangements in the seminiferous tubule; A-single (A<sup>s</sup> ), A-paired (Apr), and A-aligned (Aal). Division of As spermatogonia leads either to produce individual two new cells (self-renewing cells) or connected two cells by intercytoplasmic bridges (Apr). Further divisions of Apr lead to formation of Aal or chains of 4, 8, 16, and occasionally 32 cells. A<sup>s</sup> represents the stem cell pool and same characteristics may remain among few Apr cells as well. Larger chains of Aal (8, 16, 32) differentiate toward the Type A1 spermatogonia and then give rise to A2, A3, A4, Intermediate and B, respectively. These differentiated spermatogonia divide in a synchronize manner and found at specific stages of the seminiferous epithelial cycle. B spermatogonia differentiate into spermatocytes, and they undergo further divisions by meiosis to produce secondary spermatocytes and haploid spermatids, respectively. Single Apr cell passes eight mitotic steps resulting 1024 spermatocytes, and total 4096 haploid spermatids from subsequent meiotic division. Spermatids are subjected to 16 steps of morphological changes to become mature spermatozoa [7–9].

There are two other models to describe SSCs self-renewal: A0/A1 model and A-dark and A-pale model. In A0/A1 model normal spermatogenesis is maintained by an "active" pool of SSCs (A1) and other quiescent "reserve" pool of SSCs (A0) is mobilized only following an insult to spermatogenesis [10]. In higher primates and humans two types of morphologically distinct SSCs are described, Adark and Apale. Observing biological functions of two cell types, Apale is considered as progenitor cells and Adark as true stem cells. Adark represents only 1% of spermatogonia population, and stay dormant or divide very rarely if only progenitor cells have been destroyed. Apale proliferate at defined periods during each cycle of the seminiferous epithelium and differentiate into B spermatogonia while leaving sufficient amount Apale as functional reserve. In primates, single Apale involve 5 mitotic divisions producing 32 spermatocytes and finally 128 spermatids. Amount of clonal expansion is very low in humans, and only 16 haploid cells are produced through 2 mitotic and meiotic divisions as depicted in **Figure 1** [11]. Due to low number of haploid cells produced by a single cell, both humans and primates maintain a population of progenitor cells (Apale) as a replenishment reserve. This is to minimize mitotic activity of true stem cells and preserve their genetic stability. Thus, the role of SSCs is to regenerate and sustain a cycling cell lineage, while progenitor population which is lacking regenerative capacity contributes to steady-state conditions [12]. There is no consensus on SSCs self-renewal in aforementioned models; whether it is through symmetrical (produce two stem cells or two interconnected cells destined to differentiate) or asymmetrical (produce one stem cell and other cell committed to differentiate). Using a mouse model, Wu


In normal seminiferous epithelium, there is a well balance between SSCs self-renewal and differentiation. Loss of the equilibriums may cause either germ cell tumor or infertility subsequent to SSCs depletion. However, in specific situations, such as toxicity-induced spermatocytes destruction, this balance may be shifted toward differentiation over proliferation. One of the main regulators identified in the SSCs self-renewal is glial cell line-derived neurotrophic factor (GDNF) secreted by Sertoli cells [24]; whereas, well-known differentiation factors are stem cell factor (SCF) secreted by Sertoli cells and biologically active derivative of vitamin A; retinoic acid (RA).

*In Vitro* Spermatogenesis; Past, Present, and Future http://dx.doi.org/10.5772/intechopen.73505 29

Knowledge on cytological markers expressed or suppressed at different stages of spermatogenesis is a key factor on rapid development of *in vitro* spermatogenesis strategies. Several cytological markers such as genes, transcription factors, cytokines, growth factors, enzymes, other proteins and micro RNAs (miRNAs), etc., have been studied at distinct phases of spermatogenesis pathway from embryonic germ cells allocation to postnatal spermatogenesis process which is broadly divided into three phases; proliferation, differentiation, and spermiogenesis. Some of them are surface antigens found in different regions of the sperm and others are intracellular; in the nucleus or cytoplasm. Knowledge on the expression of stagespecific cytological markers is vital on studying germ cells biology, normal and abnormal pathways of spermatogenesis and deciding corrective measures. Proper process of spermatogenesis requires precise coordination of multitude of genes. Stage-specific surface markers may be involved in differentiation process at least part by interaction with Sertoli cells [25]. Surface antigens are extensively used for tracking subset of germ cells at specific differentiation stages, but the process is hampered as very few markers have been identified so far. Although most of these markers expressed on SSCs and early stages of differentiation, little or none of these antigens remaining on the head or tail of the sperm [26]. Majority of markers have characterized with animal germ cells, specifically using rat and mouse germ cells, and very few of them have tested in humans. We assume that the presence and behavior of major-

ity of these genes are more or less similar between animals and humans.

The chemokine receptor type 4 receptor (*CXCR4*) and *DDX4* or mouse vasa homolog (*MVH*) genes are first expressed during migratory phase of PGCs, and *MVH* expression is continued until post-meiotic germ cells are formed. Decreased proliferative capacity of PGCs and defective spermatogenesis has been observed in *MVH* null mice. *PIWI*, *Fragilis*, *SSEA1*, and *STELLA* are other genes expressed in different levels in migratory PGSc. *Fragilis* is considered to be important for the migration of PGCs toward the genital ridges [27, 28]. B-lymphocyte-induced maturation protein-1 gene (*BLIMP1*) is involved in the initial specification of PGCs. Germ cells positive for *BLIMP1* proliferate continuously, and this process can be helpful to express other PGCs markers such as *Fragilis* and *STELLA*. A network of transcription factors are involved in maintaining embryonic properties of stem cells. *SALL4* is a member of Spalt-like transcription factor family, highly expressed in multiple embryonic tissues including PGCs and gonocytes. It is also involved in SPCs differentiation. Promyelocytic Leukemia Zinc Finger (*PLZF* or

*2.3.1. PGCs, SSCs, and spermatogonial progenitor cells (SPCs) markers*

**2.3. Markers expression during spermatogenesis**

**Figure 1.** Pre-meiotic steps of spermatogenesis (SSCs to pre-leptotene spermatocytes) in different species of mammals. Spermatogonial stem cells Undifferentiated spermatogonia *Progenitor cells*. Int – intermediate, Spc - spermatocytes.

et al. support the theory of asymmetrical division. Furthermore, they have proposed that fate decision of mammalian SSCs bifurcation is autonomous and stochastic [13].

Spermiogenesis is the process of transformation of spherical, haploid spermatids (n) to sperm-like mature spermatids. Human spermatid develops into a mature sperm through a series of 12 steps and it takes about 5 weeks. It is assumed that nuclear condensation during this process shuts RNA synthesis, and proteins required in the period (mainly protamine) are produced by stored mRNAs derived from the diploid phase of spermatogenesis [14]. Contrary to this suggestion, supportive evidences are emerging on the minor activity of transcription in haploid spermatids as well [15]. Spermiation is the last process involving breakage of the structures and bonds anchoring mature spermatids to Sertoli cells in order to release spermatozoa into the tubule lumen. Peristaltic waves created by peritubular smooth muscle cells help to move spermatozoa and testicular fluids through the seminiferous tubules to the epididymis [16]. This ~10–16 days migration through epididymis helps sperm to attain motility and natural fertilization capacity up to a certain extent [17, 18]. The total motility and fertilizability is gained only after mixing with accessory sex gland secretions [19].

#### **2.2. Regulatory mechanisms of natural spermatogenesis**

Number of sperm produced per day by testes (daily sperm production, DSP) is a tool for quantitative assessment of spermatogenesis. DSP can decrease with reduced amount of true stem cells present, failure to produce committed Apale cells, changes in niche environment due to multitude of causes, age (DSP is low in very young and older men), etc. [20]. However, even in the normal spermatogenic procedure germ cells may degenerate at various levels; preleptotene and leptotene spermatocytes in older men, and pachytene/diplotene spermatocytes across all ages. This would be a mechanism of eliminating cells with genetic abnormalities [21]. Other possible reason is to maintain the ratio of Sertoli cells to germ cells, as one Sertoli cell can assist only to a specific number of cells. Furthermore, there is no fine regulation of formation of spermatocytes in different areas of tubules, resulting unequal distribution of those cells. The apoptosis mainly involving the *BCL-2* family of apoptosis regulating proteins helps to maintain an equal density of spermatocytes along the seminiferous tubule [22, 23].

In normal seminiferous epithelium, there is a well balance between SSCs self-renewal and differentiation. Loss of the equilibriums may cause either germ cell tumor or infertility subsequent to SSCs depletion. However, in specific situations, such as toxicity-induced spermatocytes destruction, this balance may be shifted toward differentiation over proliferation. One of the main regulators identified in the SSCs self-renewal is glial cell line-derived neurotrophic factor (GDNF) secreted by Sertoli cells [24]; whereas, well-known differentiation factors are stem cell factor (SCF) secreted by Sertoli cells and biologically active derivative of vitamin A; retinoic acid (RA).

#### **2.3. Markers expression during spermatogenesis**

et al. support the theory of asymmetrical division. Furthermore, they have proposed that fate

**Figure 1.** Pre-meiotic steps of spermatogenesis (SSCs to pre-leptotene spermatocytes) in different species of mammals. Spermatogonial stem cells Undifferentiated spermatogonia *Progenitor cells*. Int – intermediate, Spc - spermatocytes.

Spermiogenesis is the process of transformation of spherical, haploid spermatids (n) to sperm-like mature spermatids. Human spermatid develops into a mature sperm through a series of 12 steps and it takes about 5 weeks. It is assumed that nuclear condensation during this process shuts RNA synthesis, and proteins required in the period (mainly protamine) are produced by stored mRNAs derived from the diploid phase of spermatogenesis [14]. Contrary to this suggestion, supportive evidences are emerging on the minor activity of transcription in haploid spermatids as well [15]. Spermiation is the last process involving breakage of the structures and bonds anchoring mature spermatids to Sertoli cells in order to release spermatozoa into the tubule lumen. Peristaltic waves created by peritubular smooth muscle cells help to move spermatozoa and testicular fluids through the seminiferous tubules to the epididymis [16]. This ~10–16 days migration through epididymis helps sperm to attain motility and natural fertilization capacity up to a certain extent [17, 18]. The total motility and

decision of mammalian SSCs bifurcation is autonomous and stochastic [13].

28 Spermatozoa - Facts and Perspectives

fertilizability is gained only after mixing with accessory sex gland secretions [19].

Number of sperm produced per day by testes (daily sperm production, DSP) is a tool for quantitative assessment of spermatogenesis. DSP can decrease with reduced amount of true stem cells present, failure to produce committed Apale cells, changes in niche environment due to multitude of causes, age (DSP is low in very young and older men), etc. [20]. However, even in the normal spermatogenic procedure germ cells may degenerate at various levels; preleptotene and leptotene spermatocytes in older men, and pachytene/diplotene spermatocytes across all ages. This would be a mechanism of eliminating cells with genetic abnormalities [21]. Other possible reason is to maintain the ratio of Sertoli cells to germ cells, as one Sertoli cell can assist only to a specific number of cells. Furthermore, there is no fine regulation of formation of spermatocytes in different areas of tubules, resulting unequal distribution of those cells. The apoptosis mainly involving the *BCL-2* family of apoptosis regulating proteins helps to maintain an equal density of spermatocytes along the seminiferous tubule [22, 23].

**2.2. Regulatory mechanisms of natural spermatogenesis**

Knowledge on cytological markers expressed or suppressed at different stages of spermatogenesis is a key factor on rapid development of *in vitro* spermatogenesis strategies. Several cytological markers such as genes, transcription factors, cytokines, growth factors, enzymes, other proteins and micro RNAs (miRNAs), etc., have been studied at distinct phases of spermatogenesis pathway from embryonic germ cells allocation to postnatal spermatogenesis process which is broadly divided into three phases; proliferation, differentiation, and spermiogenesis. Some of them are surface antigens found in different regions of the sperm and others are intracellular; in the nucleus or cytoplasm. Knowledge on the expression of stagespecific cytological markers is vital on studying germ cells biology, normal and abnormal pathways of spermatogenesis and deciding corrective measures. Proper process of spermatogenesis requires precise coordination of multitude of genes. Stage-specific surface markers may be involved in differentiation process at least part by interaction with Sertoli cells [25]. Surface antigens are extensively used for tracking subset of germ cells at specific differentiation stages, but the process is hampered as very few markers have been identified so far. Although most of these markers expressed on SSCs and early stages of differentiation, little or none of these antigens remaining on the head or tail of the sperm [26]. Majority of markers have characterized with animal germ cells, specifically using rat and mouse germ cells, and very few of them have tested in humans. We assume that the presence and behavior of majority of these genes are more or less similar between animals and humans.

#### *2.3.1. PGCs, SSCs, and spermatogonial progenitor cells (SPCs) markers*

The chemokine receptor type 4 receptor (*CXCR4*) and *DDX4* or mouse vasa homolog (*MVH*) genes are first expressed during migratory phase of PGCs, and *MVH* expression is continued until post-meiotic germ cells are formed. Decreased proliferative capacity of PGCs and defective spermatogenesis has been observed in *MVH* null mice. *PIWI*, *Fragilis*, *SSEA1*, and *STELLA* are other genes expressed in different levels in migratory PGSc. *Fragilis* is considered to be important for the migration of PGCs toward the genital ridges [27, 28]. B-lymphocyte-induced maturation protein-1 gene (*BLIMP1*) is involved in the initial specification of PGCs. Germ cells positive for *BLIMP1* proliferate continuously, and this process can be helpful to express other PGCs markers such as *Fragilis* and *STELLA*. A network of transcription factors are involved in maintaining embryonic properties of stem cells. *SALL4* is a member of Spalt-like transcription factor family, highly expressed in multiple embryonic tissues including PGCs and gonocytes. It is also involved in SPCs differentiation. Promyelocytic Leukemia Zinc Finger (*PLZF* or *ZBTB16*) shows lower expression levels in embryonic germ cells and its peak in postnatal SPCs. It helps to maintain the properties of SPCs and also detected in early stages of SSCs differentiation. *SALL4* and *PLZF* physically interact and mutually oppose one another's localization to cognate chromatin domains depending on their relative expression levels at distinct stages of germ cells development. c-KIT, the transmembrane tyrosine kinase receptor for stem cell factor (SCF), also known as KIT ligand (KL) is essential to the proliferation and survival of differentiating spermatogonia, and its expression directly repressed by *PLZF*. At the time of differentiation, *SALL4* level increases with suppression of *PLZF* facilitating to expression of *c-KIT* [29]. *GPR125*, an orphan adhesion-type G-protein-coupled receptor, is another gene exclusively expressed in SPCs and SSCs, and not in differentiated spermatocytes. *GPR125* positive cells can be cultured in undifferentiated state with remarkable increase in their number [30]. A gene named transcriptional repressor inhibitor of differentiation 4 (*ID4*) was recently identified using a transgenic mouse model and it was highly expressed in most gonocytes, a subpopulation of SSCs, and a minor subset of pachytene spermatocytes [12]. But, they conclude the appearance of *ID4* in pachytene stage may be nonspecific, and proposed that *ID4* positive subpopulation may be a heterogeneous population of SPCs and SSCs in mouse. Another subset of rare and highly proliferative Asingle spermatogonia has been characterized in mouse by expression of the paired box transcription factor (*PAX7*). Using cell lineage tracing studies they have confirmed that *PAX7* cells function as bona fide stem cells [31]. The marker is coexpressed with well characterized other spermatogonial markers such as, c-*KIT, PLZF, FOXO 1, RET*, and *GFRα1. PAX7* was reported perfectly conserved in 11 different species, and it is resistant to chemo and radiotherapy insults [32]. GDNF seems to stimulate SSCs self-renewal by signaling through *Ret* and *GFRα1* receptors system, and overexpression causes to an increase of undifferentiated spermatogonia in the testis [24]. POU family transcription factor 1 or octamer-binding transforming factor4 (*POU5F1/OCT4*) is expressed throughout the PGCs migration and later on in SSCs and differentiation male germ cells up to pachytene stage [5]. *Oct4* is rather considered as a pluripotency marker and expression is inhibited when RA binds to the responsive site, allowing the cells for differentiation. Many other markers expressed by SSCs and SPCs have described such as, *TERT, POU3F1, RBM, HSP90x, NGN3, NANOS2* & *3, SOHLH1* & *2,* integrin alpha chain 6 (*ITGA6 /α6-Integrin/CD49f*), *LIN28*, *UTF1*, *CDH1*, *ITGB1 (β1-Integrin/CD29*), *EPCAM* (*CD326*), *CD9*, *CD24* and *THY1* (*CD90*). *LIN28* and *EPCAM* are increasingly expressed in malignant germ cell tumors indicating their role as maintenance of cells in undifferentiated state. The *NANOS2* is also reported to block germ cells differentiation and lack of the gene induces progressive loss of germ cells in the postnatal testis [24, 28, 33–35]. New four marker genes specifically found in mouse PGCs and SSCs, but not in somatic cells were described as, *FKBP6, MOV10l1, 4930432K21Rik,* and *TEX13* recently [36].

of SSCs in humans. SSCs expressing, epithelial cell adhesion molecule (*EPCAM*), *THY1* and *ITGA6* have been enriched using fluorescence-activated cell sorting (FACS) and magnetic-

*In Vitro* Spermatogenesis; Past, Present, and Future http://dx.doi.org/10.5772/intechopen.73505 31

These markers can be categorized according to the timing of meiotic cycle; pre-meiotic, mei-

*c-KIT* is an early differentiation marker highly expressed in Aal spermatocytes onward. c-KIT and its ligand SCF or KL are involved in growth and survival of germ cells. Interaction between the *SCF* positive Sertoli cells and the *c-KIT* positive germ cells are helpful for the progression through meiosis. Cytochrome p450 family 26, sub family b, polypeptide1 (*CYP26b1*) gene is increasingly expressed in pre-meiotic germ cells. It may prevent spermatocytes enter into meiosis by blocking the action RA, which is one of the key signaling molecule that helps to induce meiosis by binding through nuclear RA receptors. Stimulated by retinoic acid 8 (*STRA 8*) is the major responsive gene for RA induction and prominently expressed in pre-meiotic cells. At the same time RA receptors are expressed from type A spermatogonia to pre-leptotene spermatocytes in mice. Similarly, growth factor KL increases the percentage of meiotic entry in cultured spermatogonia concomitantly with an upregulation of *STRA8*. Activation of phosphatidyl inositol 3 kinase (*PI3K*) signaling appears to be important in meiotic initiation of germ cells [33, 42, 43]. XT-1 is an adhesion related surface antigen found on differentiation spermatocytes in mouse testes. It is first detectable and uniformly distributed on leptotene spermatocytes, and later localized on the base of the head, tail and cytoplasmic lobe of the elongating spermatids [26]. Bone morphogenetic protein4 (BMP4) is an early differentiation marker, mainly activate through cell adhesion pathways and also upregulate c-*KIT* expression. It is prominently expressed in pachytene stage spermatocytes, and downstream proteins, SMAD1, 5, 8 are phosphorylated during BMP4-induced differentiation [17, 33]. SMAD1, 4, and 5 proteins are found in SSCs to round spermatids, while SMAD7 is found in differentiating spermatogonia up to round spermatids. SMAD8 is detected from spermatocytes to elongating spermatids [44]. Deleted in azoospermialike gene (*DAZL*)*, VASA, BLIMP1, STELLA* are also considered as pre-meiotic early germ cells differentiation markers [34]. Highest expression of *DAZL* is found in pachytene spermatocytes, and is assisted in the translation of MVH in the males. *Cyclin D2* is another gene expressed

around epithelial stage VIII when the Aal spermatogonia differentiate into A<sup>1</sup>

Specific markers well defined for meiotic germ cells are synaptonemal complex protein 3 (*SCP3*) gene and dosage suppressor of mck1 homolog (*DMC1*). Both SCP2 and SCP3 are components of the lateral element of the synaptonemal complexes and are associated with the centromeres in meiotic metaphase I cells. Another antigen named testicular differentiation antigen 95 (TDA95), residing on zygotene and early pachytene spermatocytes has been defined using two monoclonal antibodies, CA12 and BC7. TDA95 may be one of the cell adhesion molecules between

stage [23].

activated cell sorting (MACS) techniques [38–41].

*2.3.2. Differentiation markers*

*2.3.2.1. Pre-meiotic*

*2.3.2.2. Meiotic*

otic, and post-meiotic markers.

The above markers have been described using different techniques and most of them may have conserved among closely related animals during evolution. Results from RT-PCR analysis of freshly isolated human spermatogonia indicated that they are positive for *GPR125*, *GFRα1*, *PLZF*, ubiquitin carboxyl-terminal esterase L1 (*UCHL1*), and *RET* transcripts [37]. Using immunofluorescence and colorimetric staining it has been shown that human spermatogonia on the basement membrane express *UTF1*, *SALL4*, *ZBTB16*, *GFRα1*, *UCHL1*, *GPR125*, *LIN28*, *EXOSC10*, *FGFR3*, *DSG2*, *CBL*, *SSX2*, *OCT2*, *OCT4a/b*, *TERT*, *NANOG*, *ENO2,* and *PCNA* (a proliferation marker). Not like in rodents, *GPR125* is expressed only in subset of SSCs in humans. SSCs expressing, epithelial cell adhesion molecule (*EPCAM*), *THY1* and *ITGA6* have been enriched using fluorescence-activated cell sorting (FACS) and magneticactivated cell sorting (MACS) techniques [38–41].

#### *2.3.2. Differentiation markers*

These markers can be categorized according to the timing of meiotic cycle; pre-meiotic, meiotic, and post-meiotic markers.

#### *2.3.2.1. Pre-meiotic*

*ZBTB16*) shows lower expression levels in embryonic germ cells and its peak in postnatal SPCs. It helps to maintain the properties of SPCs and also detected in early stages of SSCs differentiation. *SALL4* and *PLZF* physically interact and mutually oppose one another's localization to cognate chromatin domains depending on their relative expression levels at distinct stages of germ cells development. c-KIT, the transmembrane tyrosine kinase receptor for stem cell factor (SCF), also known as KIT ligand (KL) is essential to the proliferation and survival of differentiating spermatogonia, and its expression directly repressed by *PLZF*. At the time of differentiation, *SALL4* level increases with suppression of *PLZF* facilitating to expression of *c-KIT* [29]. *GPR125*, an orphan adhesion-type G-protein-coupled receptor, is another gene exclusively expressed in SPCs and SSCs, and not in differentiated spermatocytes. *GPR125* positive cells can be cultured in undifferentiated state with remarkable increase in their number [30]. A gene named transcriptional repressor inhibitor of differentiation 4 (*ID4*) was recently identified using a transgenic mouse model and it was highly expressed in most gonocytes, a subpopulation of SSCs, and a minor subset of pachytene spermatocytes [12]. But, they conclude the appearance of *ID4* in pachytene stage may be nonspecific, and proposed that *ID4* positive subpopulation may be a heterogeneous population of SPCs and SSCs in mouse. Another subset of rare and highly proliferative Asingle spermatogonia has been characterized in mouse by expression of the paired box transcription factor (*PAX7*). Using cell lineage tracing studies they have confirmed that *PAX7* cells function as bona fide stem cells [31]. The marker is coexpressed with well characterized other spermatogonial markers such as, c-*KIT, PLZF, FOXO 1, RET*, and *GFRα1. PAX7* was reported perfectly conserved in 11 different species, and it is resistant to chemo and radiotherapy insults [32]. GDNF seems to stimulate SSCs self-renewal by signaling through *Ret* and *GFRα1* receptors system, and overexpression causes to an increase of undifferentiated spermatogonia in the testis [24]. POU family transcription factor 1 or octamer-binding transforming factor4 (*POU5F1/OCT4*) is expressed throughout the PGCs migration and later on in SSCs and differentiation male germ cells up to pachytene stage [5]. *Oct4* is rather considered as a pluripotency marker and expression is inhibited when RA binds to the responsive site, allowing the cells for differentiation. Many other markers expressed by SSCs and SPCs have described such as, *TERT, POU3F1, RBM, HSP90x, NGN3, NANOS2* & *3, SOHLH1* & *2,* integrin alpha chain 6 (*ITGA6 /α6-Integrin/CD49f*), *LIN28*, *UTF1*, *CDH1*, *ITGB1 (β1-Integrin/CD29*), *EPCAM* (*CD326*), *CD9*, *CD24* and *THY1* (*CD90*). *LIN28* and *EPCAM* are increasingly expressed in malignant germ cell tumors indicating their role as maintenance of cells in undifferentiated state. The *NANOS2* is also reported to block germ cells differentiation and lack of the gene induces progressive loss of germ cells in the postnatal testis [24, 28, 33–35]. New four marker genes specifically found in mouse PGCs and SSCs, but not in somatic cells

30 Spermatozoa - Facts and Perspectives

were described as, *FKBP6, MOV10l1, 4930432K21Rik,* and *TEX13* recently [36].

The above markers have been described using different techniques and most of them may have conserved among closely related animals during evolution. Results from RT-PCR analysis of freshly isolated human spermatogonia indicated that they are positive for *GPR125*, *GFRα1*, *PLZF*, ubiquitin carboxyl-terminal esterase L1 (*UCHL1*), and *RET* transcripts [37]. Using immunofluorescence and colorimetric staining it has been shown that human spermatogonia on the basement membrane express *UTF1*, *SALL4*, *ZBTB16*, *GFRα1*, *UCHL1*, *GPR125*, *LIN28*, *EXOSC10*, *FGFR3*, *DSG2*, *CBL*, *SSX2*, *OCT2*, *OCT4a/b*, *TERT*, *NANOG*, *ENO2,* and *PCNA* (a proliferation marker). Not like in rodents, *GPR125* is expressed only in subset

*c-KIT* is an early differentiation marker highly expressed in Aal spermatocytes onward. c-KIT and its ligand SCF or KL are involved in growth and survival of germ cells. Interaction between the *SCF* positive Sertoli cells and the *c-KIT* positive germ cells are helpful for the progression through meiosis. Cytochrome p450 family 26, sub family b, polypeptide1 (*CYP26b1*) gene is increasingly expressed in pre-meiotic germ cells. It may prevent spermatocytes enter into meiosis by blocking the action RA, which is one of the key signaling molecule that helps to induce meiosis by binding through nuclear RA receptors. Stimulated by retinoic acid 8 (*STRA 8*) is the major responsive gene for RA induction and prominently expressed in pre-meiotic cells. At the same time RA receptors are expressed from type A spermatogonia to pre-leptotene spermatocytes in mice. Similarly, growth factor KL increases the percentage of meiotic entry in cultured spermatogonia concomitantly with an upregulation of *STRA8*. Activation of phosphatidyl inositol 3 kinase (*PI3K*) signaling appears to be important in meiotic initiation of germ cells [33, 42, 43].

XT-1 is an adhesion related surface antigen found on differentiation spermatocytes in mouse testes. It is first detectable and uniformly distributed on leptotene spermatocytes, and later localized on the base of the head, tail and cytoplasmic lobe of the elongating spermatids [26]. Bone morphogenetic protein4 (BMP4) is an early differentiation marker, mainly activate through cell adhesion pathways and also upregulate c-*KIT* expression. It is prominently expressed in pachytene stage spermatocytes, and downstream proteins, SMAD1, 5, 8 are phosphorylated during BMP4-induced differentiation [17, 33]. SMAD1, 4, and 5 proteins are found in SSCs to round spermatids, while SMAD7 is found in differentiating spermatogonia up to round spermatids. SMAD8 is detected from spermatocytes to elongating spermatids [44]. Deleted in azoospermialike gene (*DAZL*)*, VASA, BLIMP1, STELLA* are also considered as pre-meiotic early germ cells differentiation markers [34]. Highest expression of *DAZL* is found in pachytene spermatocytes, and is assisted in the translation of MVH in the males. *Cyclin D2* is another gene expressed around epithelial stage VIII when the Aal spermatogonia differentiate into A<sup>1</sup> stage [23].

#### *2.3.2.2. Meiotic*

Specific markers well defined for meiotic germ cells are synaptonemal complex protein 3 (*SCP3*) gene and dosage suppressor of mck1 homolog (*DMC1*). Both SCP2 and SCP3 are components of the lateral element of the synaptonemal complexes and are associated with the centromeres in meiotic metaphase I cells. Another antigen named testicular differentiation antigen 95 (TDA95), residing on zygotene and early pachytene spermatocytes has been defined using two monoclonal antibodies, CA12 and BC7. TDA95 may be one of the cell adhesion molecules between

is a candidate meiotic regulator gene conserved among different species from drosophila to human, and deficient animals are infertile due to meiotic arrest in their male germ cells. The protein is detectable from pro-metaphase of first meiotic division to diplotene spermatocytes in humans and up to early spermatids in mice [45]. Similar expression pattern showed another meiotic promoter protein CDC25A, which was found in 1ry and 11ry spermatocytes and in some species up to elongating spermatids. Variations of three different isoforms of *BOULE* (*BOULE1, 2,* & *3*) during spermatogenesis were described in humans [46]. *SPO11* (a type II topoisomer-

*In Vitro* Spermatogenesis; Past, Present, and Future http://dx.doi.org/10.5772/intechopen.73505 33

Expression of acrosin (*ACR*), transition protein 1 & 2 (*TNP1, 2*), ubiquitin-activating enzyme (*UBE1Y*), Kinesin light chain proteins (*KLC3*) and protamine1 & 2 (*PRM1, 2*) genes has been reported in post-meiotic cells [37]. *ACR* gene is transcribed in the diploid stage, and translationally expressed in the post-meiotic haploid cells. Hence, proacrosin is first localized in the cytoplasm of round spermatids. PRM1 and PRM2 are small and highly basic proteins that are reported to be transcribed in round and elongating spermatids. KLC3 is a testis specific protein and acts as an anchor protein for binding mitochondria to outer dense fibers of sperm tail. During the spermiogenesis, somatic histones are replaced with testis specific nuclear proteins, termed as transition proteins (TNP1 & TNP2), and subsequently TNPs are replaced with PRMs. Formation of extensive disulfide cross links between PRMs results in condensation of nuclear proteins and repression of transcriptional activity [48]. Alteration of *PRM* expression has been reported to affect human male fertility. For example, high susceptibility to DNA damage was reported with diminished levels [49]. Bone morphogenetic family proteins BMP8a and BMP8b receptors are expressed in male germ cells in a bimodal manner. First, low levels of transcripts are found in spermatogonia and primary spermatocytes, and subsequently higher levels are expressed in round spermatids. BMP8b is most important in initiation and maintenance of spermatogenesis [50]. **Figure 2** depicts stage-specific expression

**3. Attempts to differentiate spermatozoa outside the niche (***in vitro***,** 

Understanding the components of SSCs niche and their interactions with each other are vital aspects in regeneration of spermatogenesis *in vitro*. The *in vivo* niche of mammalian SSCs is comprised of Sertoli cells, peritubular cells, and a complex array of matrix proteins. The normal SSC pool is maintained throughout adulthood, through signals provided by adhesion molecules and other cell surface receptors. SSCs are exposed to signals from both tubular lumen and the interstitial space sides of the basement membrane. Fate of SSCs is regulated mainly by Sertoli cells, inter-tubular blood vessels, and surrounding Leydig cells also have a role [51]. Steady state of germ cell niche can be disturbed by physiological changes of individual components by intrinsic or external factors. Regeneration of sperm has been difficult due to incomplete understanding of complex interactions within the niche environment, and germ cell-specific events such as, meiosis, chromatin re-modeling/repackaging,

ase), *H2A*, *TH2b* are other few genes, needed for meiotic recombination [43, 47].

of few selected markers commonly found in literature.

*in vivo,* **and** *ex vivo***)**

*2.3.2.3. Post-meiotic*

**Figure 2.** Stage-specific germ cell markers compiled from different sources. Most of them are germ cell specific and some of the markers are common to both germ cell and few types of somatic lineage cells. These markers are significant in solid identification of germ cells at different phases of development.

spermatocytes and Sertoli cells, and plays an essential role during early meiotic prophase of spermatogenesis. *SCP1, CREST,* and *Tesmin* are other markers present in human pachytene spermatocytes. *Tesmin* expression coincides with meiotic entry of germ cells [25, 37]. *BOULE*

is a candidate meiotic regulator gene conserved among different species from drosophila to human, and deficient animals are infertile due to meiotic arrest in their male germ cells. The protein is detectable from pro-metaphase of first meiotic division to diplotene spermatocytes in humans and up to early spermatids in mice [45]. Similar expression pattern showed another meiotic promoter protein CDC25A, which was found in 1ry and 11ry spermatocytes and in some species up to elongating spermatids. Variations of three different isoforms of *BOULE* (*BOULE1, 2,* & *3*) during spermatogenesis were described in humans [46]. *SPO11* (a type II topoisomerase), *H2A*, *TH2b* are other few genes, needed for meiotic recombination [43, 47].

#### *2.3.2.3. Post-meiotic*

spermatocytes and Sertoli cells, and plays an essential role during early meiotic prophase of spermatogenesis. *SCP1, CREST,* and *Tesmin* are other markers present in human pachytene spermatocytes. *Tesmin* expression coincides with meiotic entry of germ cells [25, 37]. *BOULE*

**Figure 2.** Stage-specific germ cell markers compiled from different sources. Most of them are germ cell specific and some of the markers are common to both germ cell and few types of somatic lineage cells. These markers are significant in

solid identification of germ cells at different phases of development.

32 Spermatozoa - Facts and Perspectives

Expression of acrosin (*ACR*), transition protein 1 & 2 (*TNP1, 2*), ubiquitin-activating enzyme (*UBE1Y*), Kinesin light chain proteins (*KLC3*) and protamine1 & 2 (*PRM1, 2*) genes has been reported in post-meiotic cells [37]. *ACR* gene is transcribed in the diploid stage, and translationally expressed in the post-meiotic haploid cells. Hence, proacrosin is first localized in the cytoplasm of round spermatids. PRM1 and PRM2 are small and highly basic proteins that are reported to be transcribed in round and elongating spermatids. KLC3 is a testis specific protein and acts as an anchor protein for binding mitochondria to outer dense fibers of sperm tail. During the spermiogenesis, somatic histones are replaced with testis specific nuclear proteins, termed as transition proteins (TNP1 & TNP2), and subsequently TNPs are replaced with PRMs. Formation of extensive disulfide cross links between PRMs results in condensation of nuclear proteins and repression of transcriptional activity [48]. Alteration of *PRM* expression has been reported to affect human male fertility. For example, high susceptibility to DNA damage was reported with diminished levels [49]. Bone morphogenetic family proteins BMP8a and BMP8b receptors are expressed in male germ cells in a bimodal manner. First, low levels of transcripts are found in spermatogonia and primary spermatocytes, and subsequently higher levels are expressed in round spermatids. BMP8b is most important in initiation and maintenance of spermatogenesis [50]. **Figure 2** depicts stage-specific expression of few selected markers commonly found in literature.
