Where Do Adult Leydig Cells Come From?

Testosterone-producing adult Leydig cells emerge at puberty, and most persist thereafter without turning over. But where do the adult Leydig cells come from and how is the process regulated? Moreover, if adult Leydig cells are first seen at puberty, what accounts for the high testosterone production in the fetal and perinatal periods? Two separate Leydig cell generations have been shown to develop successively in the testis between embryogenesis and puberty. In rats, the first of these, the fetal Leydig cells (FLCs), differentiate during gestation from mesenchymal cells situated between the testis cords. The FLCs produce high levels of testosterone in the fetus and neonate. Testicular descent, prostate and seminal vesicle growth, and maturation of the external genitalia are all dependent on this pubertal androgen surge [1]. After birth, however, most of the FLCs dedifferentiate or undergo apoptosis, to be replaced by cells that enter the adult Leydig cell lineage. The origin of the FLCs remains uncertain, though it seems clear that they derive from stem cells [2, 3]. Although previously suspected, the paper by Griffin et al. [4] provides direct evidence that the steroidogenic function of FLCs is independent of luteinizing hormone (LH). Exactly how FLC steroidogenic function is regulated is unclear.

If, as widely reported, fetal Leydig cells do not give rise to the adult Leydig cell population, where do the adult cells come from? In a series of elegant studies, Matthew Hardy and his colleagues demonstrated that three separate transitions of cells are involved in the development and differentiation of adult Leydig cells, with the ultimate source of these cells being a pool of undifferentiated, self-renewing stem cells [5–9]. The sequence of cellular events is illustrated in Figure 1. Hardy and his colleagues showed that there were spindle-shaped cells in the testicular interstitium at 1 wk postpartum that were HSD3B-negative and, therefore, had not yet entered the Leydig cell lineage; that the culture of these cells in media containing appropriate growth factors stimulated the proliferation of the cells over the course of 6 mo without their differentiation; and that when placed in a differentiation-inducing medium that contained a combination of thyroid hormone, insulin-like growth factor 1, and LH, about 40% of the cells expressed HSD3B and then synthesized testosterone by about a week thereafter. Moreover, when injected into a Leydig cell-depleted adult testis, the cells were able to colonize the interstitial compartment and ultimately to express HSD3B activity in vivo. These results suggested strongly that the isolated cells were stem cells.

Fig. 1.

Leydig cell differentiation: stem to adult cells. Modified from Chen et al., 2009 [10].

The differentiation of the stem Leydig cells (SLCs) to progenitor Leydig cells (PLCs), which in the rat has been shown to occur between Postnatal Days 10 and 14, signals the beginning of the Leydig cell lineage. In contrast to SLCs, the PLCs express HSD3B. Due to high levels of steroid 5-alpha-reductase and dehydrogenase/reductase (SDR family) member 9 (DHRS9, also known as 3αHSD) activity and low levels of HSD17B expressed by PLCs, androsterone is their major steroid product. The mechanism by which SLCs differentiate to PLCs is unclear. This question can and has been examined using isolated cells, but also can be addressed in vivo by global transcription profiling. As in the Griffin et al. study [4], such an approach is likely to identify candidate regulatory genes associated with the transition whose expression might cause it. By approximately Postpartum Day 28, the PLCs begin to transition into immature Leydig cells (ILCs), which by virtue of high levels of steroid 5-alpha-reductase and DHRS9 activities produce high levels of 5α-reduced androgens; these cells then divide once or twice and differentiate into testosterone-secreting adult Leydig cells. These transitions have been described most fully for the rat, but similar transitions are thought to occur in other species as well, including humans. As with the SLC to PLC transition, the mechanisms responsible for the PLC to ILC and ILC to adult Leydig cell transitions are uncertain. The use of both in vitro and in vivo approaches to address these mechanisms is highly desirable.

In sum, although lack of LH stimulation in adults results in reduced steroidogenic enzyme activities and Leydig cell atrophy, LH stimulation is unlikely to be the initial stimulus for cells to enter the Leydig cell lineage or to trigger the initial expression of Leydig cell-specific genes. Previous evidence for this comes in part from the attenuated response to gonadotropin stimulation in PLCs resulting from truncation of the LH/choriogonadotropin receptor protein (LHCGR) [11]. Studies of Gnrh1^hpg mice, which are deficient in circulating LH, indicate that LH plays a critical role in the further development of Leydig cells [12]. Moreover, studies of Lhcgr knockout mice have reported that the Leydig cell development does not progress to the adult stage and that gene expression and steroid synthesis are affected [13, 14]. The observation made in the Griffin et al. article [4] that the transcriptional effects of LH insensitivity are biphasic, with an early testosterone-independent phase and a subsequent testosterone-dependent phase, extend past studies and emphasize the feasibility of using a microarray approach to elucidate regulatory mechanisms in vivo.

Adequate production of testosterone early in development is required for normal masculinization. There is compelling evidence that environmental chemicals, by causing even subtle deficiencies in testosterone production by the fetal or neonatal testis, might be involved in developmental anomolies in the male reproductive tract in wildlife and declining sperm counts in humans [15]. It is essential that we understand fetal and neonatal testosterone production, including the cells involved and how these cells are regulated, to understand early susceptibilities to disruption. It is particularly important to understand the development and function of these cells in their complex in vivo setting.