and Molecular Biology of Spermatogenesis
Spermatogenesis starts at puberty and human beings produce 1000 spermatozoa with each heartbeat. This enormous spermatogenesis is maintained by spermatogonial stem cells (SSCs) at the basement of the ST [4]. Alkaline phosphatase positive epiblast cells of ectoderm are the embryonic origin of SSCs. About a hundred such cells, which had been lineage restricted from day 7.2 post mating, develop to primordial germ cells (PGCs) in the mouse embryo [5]. The PGCs multiply and reach the genital ridge which is prominent by day 11.5 post coitum [6]. About 10 000 unipotent PGCs are found in each mice gonad. In the gonadal ridge, PGCs harbor the seminiferous cords and become mitotically arrested at day 13.5 post coitum, thereafter termed as gonocytes or prespermatogonia or prospermatogonia. Prospermatogonia, morphologically different from PGCs, undergo extensive epigenetic reprogramming for their later functioning as male gametes [7]. Once prospermatogonia reach basement peripartum, they are morphologically and biochemically different from prospermatogonia and are termed as Aundifferentiated spermatogonia. Aundifferentiated spermatogonia proliferate but remain connected as Asingle, Apaired, and Aaligned through intercellular bridges. Intercellular bridges play a role in cell to cell communication and synchronization of these mitoses. Aaligned are syncytia of 4, 8, and 16 undifferentiated spermatogonia. Currently, no method distinguishes among different types of Aundifferentiated spermatogonia [1]. An undifferentiated spermatogonia irreversibly differentiates to A1 spermatogonia under the repeated stimulation of retinoic acid. In rodents, chain identity is maintained upon this transition and it has been estimated that 64% of Apair, 94% of Aaligned4, and 100% of Aaligned8‐16 transition to the differentiating A1 state following a retinoic acid pulse [8]. A1 spermatogonia undergo six synchronized mitoses through A2, A3, A4, intermediate (In), and B spermatogonia to form primary spermatocyte [4]. In the classical Huckins and Oakberg model, SSCs are contained within the Asingle population [9, 10]. However, one current model of SSC renewal suggests that syncytia of Apaired and Aaligned spermatogonia can break to yield SSCs [11].
All undifferentiated spermatogonia other than SSCs are called progenitors. Asingle undergo symmetric divisions forming syncytia of undifferentiating spermatogonia, or there is asymmetric division forming an SSC and a progenitor Asingle, thus maintaining steady‐state spermatogenesis [4]. Total number of Asingle in adult mouse testis is estimated at 35 000 and each adult mice testis has about 3000 SSCs, which is about 0.01% of total testis cells [12], and 0.03% of the entire testicular germ cell population. Furthermore, 33 × 104 undifferentiated spermatogonia and 280 × 104 differentiating spermatogonia populate each testis in the mouse [8]. SSCs as well as epiblast and PGCs from days 6–16.5 post coitum embryo can regenerate spermatogenesis upon transplantation to appropriate donor testes [13].
SSCs, capable of self‐renewal and generating progenitor cells through symmetrical and/or asymmetrical divisions, reside in a specific microenvironment within the ST termed as a germ line stem cell niche [14]. Within this microenvironment of the niche, SSCs are intermingled with other undifferentiated spermatogonia. Sertoli cells in communication with other somatic cells of the testes secrete factors to maintain the microenvironment of the niche. Within the niche, SSCs are located toward areas of the ST closer to interstitial vasculature [15].
Development of preleptotene spermatocyte from type B spermatogonia marks a cell’s detachment and migration away from the basement membrane. Preleptotene spermatocytes cross the tight Sertoli cell junctions and reach the adluminal portion of the ST. Primary spermatocytes first divide mitotically and then form short‐lived haploid secondary spermatocytes through meiosis I. During prophase of meiosis, preleptotene spermatocytes differentiate into leptotene, zygotene, pachytene, and diplotene, respectively. Haploid spermatids are formed by equational meiosis II of secondary spermatocytes. Round spermatids mature into spermatozoa through various steps of spermiogenesis without any increase in cell number. Spermatozoa are released into the lumen of the ST through the process of spermiation [16]. Figure 3.1 shows the steps in spermatoogenesis.
Figure 3.1 Steps in spermatogenesis.
Spermatogenesis can be divided into spermatocytogenesis, meiosis, and spermiogenesis. During spermatocytogenesis, germ cells divide by several mitoses to increase the yield of spermatogenesis, renew spermatogonial stems cells, produce more undifferentiated spermatogonia, and finally produce primary spermatocytes. During meiosis, recombination of genetic material happens, homologous chromosomes move apart, and chromosome number is reduced by half to yield haploid round spermatids. Spermiogenesis involves differentiation of haploid round spermatids into mature haploid elongated spermatozoa without mitosis or meiosis [16].
Markers to Study Spermatogenesis
Id4, Gfra1, Bcl6b, Lhx1, and Etv5 are upregulated in SSCs. Sohlh1, Ngn3, and Kit are expressed by progenitor spermatogonia [17]. THY1, ID4, and GFRA1 are markers for SSCs, NGN3 and KIT for progenitors, PLZF/ZBTB16 for all undifferentiated spermatogonia, and VASA for all germ cells [18].
Hormones and Spermatogenesis
Testosterone, follicle stimulating hormone (FSH), and luteinizing hormone (LH) are the main hormones required for spermatogenesis. Deficiency of these hormones causes germ cell apoptosis, while the administration of these suppresses apoptosis. Thus, these hormones are the survival factors required by germ cells. Under testosterone deficiency, round spermatids do not transition to elongated spermatids as the spermatids lose their attachment to Sertoli cells. FSH mainly acts indirectly through Sertoli cells. FSH has been linked to early stages in spermatogenesis, mainly spermatocytogenesis and meiosis, while testosterone is linked to later stages of spermatogenesis, mainly in spermatid differentiation and in potentiating the effect of FSH.
The Cycle of the Seminiferous Epithelium
The cycle of the seminiferous epithelium refers to is the histological fact that at a given section of the ST, different types of germ cells appear cyclically at species‐specific time intervals [16]. The notion of the cycle of the seminiferous epithelium was established toward the end of the nineteenth century [19]. Several generations of germ cells are present along the ST because spermatogenesis is a continuous process and subsequent spermatogenesis does not pause to wait for completion of the first generation to the end‐product spermatozoa and the release of these spermatozoa in the lumen of the ST [20]. Aundifferentiated spermatogonia differentiate to Adifferentiating spermatogonia (A1 and subsequent spermatogenesis) regularly after a species‐specific fixed time interval. This Aundifferentiated to A1 transition does not wait for the preceding process of “Aundifferentiated to A1 transition and finally to spermatozoon” to complete [21]. Along the section of the ST, the development of germ cells is influenced by neighboring cells, resulting in specific germ cell associations following one another in time at a given section of the ST in a perfectly organized pattern, termed the cycle of the seminiferous epithelium. This results from the fact that (i) at specific points of the ST new spermatogonia enter spermatogenesis at constant time intervals, and (ii) once the cells are engaged in spermatogenesis, their rate of differenation to the next cell type is always a constant and each step of spermatogenesis has a constant and fixed time duration. The specific germ cell associations at a given point of the ST during the cycle of the seminiferous epithelium are termed as stages, or less commonly as phases of the cycle. In the bull, the cycle is divided into eight stages and spermiation is taken as the reference point [22, 23]. In bulls, the duration of the seminiferous epithelium cycle is 13.5