Regulation of spermatogonial stem cell self-renewal in mammals

Abstract

Mammalian spermatogenesis is a classic adult stem cell-dependent process, supported by self-renewal and differentiation of spermatogonial stem cells (SSCs). Studying SSCs provides a model to better understand adult stem cell biology, and deciphering the mechanisms that control SSC functions may lead to treatment of male infertility and an understanding of the etiology of testicular germ cell tumor formation. Self-renewal of rodent SSCs is greatly influenced by the niche factor glial cell line-derived neurotrophic factor (GDNF). In mouse SSCs, GDNF activation upregulates expression of the transcription factor-encoding genes bcl6b, etv5, and lhx1, which influence SSC self-renewal. Additionally, the non-GDNF-stimulated transcription factors Plzf and Taf4b have been implicated in regulating SSC functions. Together, these molecules are part of a robust gene network controlling SSC fate decisions that may parallel the regulatory networks in other adult stem cell populations.

Figure 1

Characteristics of mammalian spermatogonial stem cell (SSC) functions and their associated niche microenvironment. (a) Two possible SSC fate decisions. Stem cells are defined by their ability to produce both more stem cells (self-renewal) and differentiating progeny (differentiation). In the mammalian testis, SSCs undergo either self-renewal, to maintain a pool of SSCs that support fertility for the majority of a male’s life span, or differentiation, resulting in the formation of A_pr spermatogonia, which are committed to the terminal pathway of spermatozoa production. (b) Possible division pathways of SSCs. Symmetrical division dictates that an SSC divides to produce either two new SSCs (self-renewal) or A_pr spermatogonia (differentiation) with an interconnecting cytoplasmic bridge; A_pr spermatogonia are destined for differentiation into spermatozoa. The asymmetrical theory suggests that SSC division produces two daughter cells: one new SSC (self-renewal) and one committed differentiating cell that produces A_pr spermatogonia upon its next division. It is currently unknown whether one or both of these pathways occur in mammalian testes. (c) Differentiation and proliferation of the spermatogonia population in rodent testes. Spermatogenesis is the sum of all germ cell divisions and transformations beginning with SSC differentiation and ending with the generation of spermatozoa. Following SSC differentiation is the formation of A_pr spermatogonia, which mitotically divide to produce A_al(4) spermatogonia followed by A_al(8) and A_al(16) spermatogonia. More mature spermatogonia are then formed and undergo another series of mitotic amplifying divisions, beginning with A₁ spermatogonia and ending with type B spermatogonia. This stage is followed by meiosis and the formation of haploid spermatozoa. (d) Postulated SSC niche microenvironment in mammalian testes. SSCs reside in the basal compartment of the seminiferous tubule, surrounded by Sertoli cells but below their tight junctions, which separate the seminiferous epithelium into basal and adluminal compartments. Stem cell niches are formed on the basis of both architectural support and specific growth factors produced by so-called niche cells. In the mammalian testis, only Sertoli cells have currently been identified as a niche cell that produces growth factors influencing SSC functions. However, contributions from other somatic cell populations surrounding the seminiferous tubules, such as peritubular myoid cells and interstitial Leydig cells, are also possible.

Figure 2

The spermatogonial stem cell (SSC) transplantation assay in mice. Stem cells are defined by their ability to reestablish the function of a tissue system from which they are derived. Spermatogenesis is a classic adult stem cell–dependent process in which SSCs continually produce terminally differentiated spermatozoa; thus, SSCs are defined by their functional ability to colonize a recipient testis and generate spermatogenesis. Currently, the only direct means to identify SSCs within a cell population and study their activity is the functional transplantation assay. (a) In this assay a testis cell population is collected fresh from a donor testis or following a culture period and microinjected into the testes of recipient males that have been depleted of germ cells with chemotoxic drugs or irradiation. Additionally, sterile mutant males that lack germ cells (e.g., W/Wv mice) can be effectively used as recipients. After a period of several months, colonies of donor-derived spermatogenesis can be detected in the recipient testes if SSCs were present in the injected cell suspension. When transgenic donors that express a marker gene (e.g., LacZ or GFP) are used, colonies of donor-derived spermatogenesis can easily be detected and quantified. Each colony is clonally derived from an individual SSC; therefore, this system provides a quantifiable measure of SSC number in an experimental cell population. In the schematic presented here, a mixed population of donor testis cells isolated from a male Rosa mouse that expresses LacZ in all germ cell types (represented as blue coloring) is microinjected into the seminiferous tubules of an immunologically compatible non-Rosa recipient testis that was pretreated with a chemotoxic drug (busulfan). SSCs in the injected cell suspension (distinguished from other Rosa testes cells by dark blue coloring) colonize the recipient seminiferous tubules and reestablish spermatogenesis. These donor SSC–derived colonies of spermatogenesis can then be visualized several months later upon incubation with X-Gal, which results in blue staining. (b) Use of the SSC transplantation method to assay the SSC content of different testis cell populations. (Left) Recipient testis injected with a donor Rosa cell suspension in which no SSC colonization occurred, indicating a lack of SSCs. (Middle) Recipient testis injected with a donor Rosa cell suspension containing a small number of SSCs, which is representative of a typical result from transplanting an unselected testis cell suspension. (Right) Recipient testis with abundant donor SSC colonization, which is indicative of results obtained following injection of an SSC-enriched cell suspension.

Figure 3

Current understanding of molecular mechanisms regulating spermatogonial stem cell (SSC) self-renewal in mice. Glial cell line–derived neurotrophic factor (GDNF) is the only growth factor demonstrated to have an essential role in regulating SSC self-renewal, and basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) enhances this influence. GDNF binds to a receptor complex consisting of c-Ret tyrosine kinase and the GPI (glycosyl phosphatidylinositol)-anchored binding molecule Gfrα1 (GDNF family receptor alpha 1). This interaction activates PI3K (phosphoinositide 3-kinase) and Src family kinase (SFK) intercellular signaling mechanisms, leading to downstream activation of Akt signaling, which influences general cellular functions such as survival and proliferation. SFK signaling also elicits a second pathway leading to the regulation of specific gene expression levels that are important for SSC self-renewal. The transcription factor–encoding genes bcl6b (B cell CLL/lymphoma 6, member B; also termed bazf), etv5 (Ets variant gene 5; also termed erm), and lhx1 (Lim homeobox protein 1; also termed lim1) are regulated through the SFK-activated pathway and are important for the maintenance of self-renewing SSC cultures. Five of the eight known mammalian SFK isoforms—c-Src (Rous sarcoma oncogene), Yes (Yamaguchi sarcoma viral oncogene), Fyn (Fyn proto-oncogene), Lyn (Lyn tyrosine kinase), and Hck (hemopoietic cell kinase)—are expressed in mouse SSCs. Additionally, observations of disrupted spermatogenesis in null mutant mice have pointed to an essential role of the transcription factors Plzf (promyelocytic leukemia zinc finger protein) and Taf4b [TATA box–binding protein (TBP)-associated factor 4b] in mouse SSC self-renewal. However, GDNF does not influence the expression of either Plzf or Taf4b in cultured SSCs, and the importance of either molecule in SSC self-renewal in vitro has not been determined. To date, mechanisms by which bFGF or EGF influences the self-renewal and survival of SSCs have not been reported.

Figure 4

Expression of transcription factors in nonpluripotent spermatogonial stem cells (SSCs) that are thought to be involved in regulating the pluripotent states of embryonic stem (ES) and induced pluripotent stem (iPS) cells. (a) Expression of Oct3/4 and Sox2 is essential for the maintenance of pluripotency in ES cells, in which these two molecules control the expression of Nanog. (b) Ectopic expression of Oct3/4, Sox2, Klf4, and Myc induces pluripotency in mouse and human fibroblasts (iPS cells). Similarly, ectopic expression of Lin28 and Nanog, in addition to expression of Oct3/4 and Sox2, also induces pluripotency of human fibroblasts. Additionally, Myc expression appears to be dispensable; iPS cells can also be generated by ectopic expression of Oct3/4, Sox2, and Klf4 alone. ES cells also express high levels of Klf4, Myc, and Lin28, but the importance of these three molecules in ES cell pluripotency has not been determined. (c) Cultured SSCs express nearly all the transcription factors regulating ES cell pluripotency and those that induce a similar potential in fibroblasts, including Oct3/4, Sox2, Klf4, Myc, and Lin28, but do not express Nanog. The absence of Nanog expression in SSCs may signify a distinct difference in the transcription factor milieu that regulates the function of an adult stem cell population such as SSCs and that of pluripotent ES and iPS cell populations. During embryo development, the first germ cells formed, primordial germ cells (PGCs), require the expression of Nanog, and these cells can become pluripotent under appropriate conditions. However, SSCs, the postnatal descendents of PGCs, do not express Nanog, and many researchers have found their conversion to pluripotency difficult. Thus, ectopic expression of Nanog may be a missing piece to the puzzle by which SSCs can be artificially transformed into a pluripotent state because they already express the array of other molecules that induce pluripotency in somatic cells.