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Biology of Reproduction 65, 660-671 (2001)
© 2001 Society for the Study of Reproduction, Inc.

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Differentiation of the Adult Leydig Cell Population in the Postnatal Testis1

S.M.L. Chamindrani Mendis-Handagama2,a,b, and H.B. Siril Ariyaratnea,b

a Departments of Comparative Medicine b Animal Science, The University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee 37996


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 LEYDIG CELL LINEAGE
 LEYDIG CELL NUMBERS
 REGULATION OF POSTNATAL...
 REFERENCES
 
Five main cell types are present in the Leydig cell lineage, namely the mesenchymal precursor cells, progenitor cells, newly formed adult Leydig cells, immature Leydig cells, and mature Leydig cells. Peritubular mesenchymal cells are the precursors to Leydig cells at the onset of Leydig cell differentiation in the prepubertal rat as well as in the adult rat during repopulation of the testis interstitium after ethane dimethane sulfonate (EDS) treatment. Leydig cell differentiation cannot be viewed as a simple process with two distinct phases as previously reported, simply because precursor cell differentiation and Leydig cell mitosis occur concurrently. During development, mesenchymal and Leydig cell numbers increase linearly with an approximate ratio of 1:2, respectively. The onset of precursor cell differentiation into progenitor cells is independent of LH; however, LH is essential for the later stages in the Leydig cell lineage to induce cell proliferation, hypertrophy, and establish the full organelle complement required for the steroidogenic function. Testosterone and estrogen are inhibitory to the onset of precursor cell differentiation, and these hormones produced by the mature Leydig cells may be of importance to inhibit further differentiation of precursor cells to Leydig cells in the adult testis to maintain a constant number of Leydig cells. Once the progenitor cells are formed, androgens are essential for the progenitor cells to differentiate into mature adult Leydig cells. Although early studies have suggested that FSH is required for the differentiation of Leydig cells, more recent studies have shown that FSH is not required in this process. Anti-Müllerian hormone has been suggested as a negative regulator in Leydig cell differentiation, and this concept needs to be further explored to confirm its validity. Insulin-like growth factor I (IGF-I) induces proliferation of immature Leydig cells and is associated with the promotion of the maturation of the immature Leydig cells into mature adult Leydig cells. Transforming growth factor {alpha} (TGF{alpha}) is a mitogen for mesenchymal precursor cells. Moreover, both TGF{alpha} and TGFß (to a lesser extent than TGF{alpha}) stimulate mitosis in Leydig cells in the presence of LH (or hCG). Platelet-derived growth factor-A is an essential factor for the differentiation of adult Leydig cells; however, details of its participation are still not known. Some cytokines secreted by the testicular macrophages are mitogenic to Leydig cells. Moreover, retarded or absence of Leydig cell development has been observed in experimental models with impaired macrophage function. Thyroid hormone is critical to trigger the onset of mesenchymal precursor cell differentiation into Leydig progenitor cells, proliferation of mesenchymal precursors, acceleration of the differentiation of mesenchymal cells into Leydig cell progenitors, and enhance the proliferation of newly formed Leydig cells in the neonatal and EDS-treated adult rat testes.

Leydig cells, luteinizing hormone, testis, testosterone, thyroid hormone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
LEYDIG CELL LINEAGE
 LEYDIG CELL NUMBERS
 REGULATION OF POSTNATAL...
 REFERENCES
 
The aim of this paper is to review the postnatal differentiation of Leydig cells in the mammalian testis. As this process has been extensively studied in rodents, most of the information presented here is from the studies of laboratory rodents. Special attention was paid to describe the characteristic features of the cells in Leydig cell lineage, precursors to Leydig cells, cell kinetics during Leydig cell differentiation, and the regulation of Leydig cell differentiation.


   LEYDIG CELL LINEAGE
TOP
 ABSTRACT
 INTRODUCTION
 LEYDIG CELL LINEAGE
LEYDIG CELL NUMBERS
 REGULATION OF POSTNATAL...
 REFERENCES
 
The populations of Leydig cells that differentiate prenatally and postnatally are identified as fetal and adult Leydig cells, respectively. The existence of these two distinct populations of Leydig cells was documented in the literature as early as 1959 [1] in the rat. Thereafter, this concept was further strengthened by the studies of Mancini et al. [2] in the human and Lording and de Kretser [3] in the rat. Early investigations on this process include many mammalian species such as the rat [3, 4] rabbit [5,6], mouse [7], pig [8], hamster [9], and ferret [10]. However, among all mammalian species, rat is the most studied species on the process of postnatal Leydig cell differentiation.

In the rat, postnatal differentiation of Leydig cells is reported to take place around the second postnatal week [1, 3]; the earliest reported is on Postnatal Day 10 [11]. This developmental process consists of multiple steps: proliferation of precursor cells, differentiation of precursor cells to Leydig cell progenitors, progenitors into newly formed adult Leydig cells, newly formed adult Leydig cells into immature adult Leydig cells, and finally maturation of the immature adult Leydig cells to mature adult Leydig cells (Fig. 1).



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  		FIG. 1. Leydig cell lineage. ALC, Adult Leydig cells

Precursor Cells

It has long been suggested that undifferentiated fibroblast-like cells or mesenchymal cells in the testis interstitium are the precursor cells to adult Leydig cells in the mammalian testis, with evidence obtained from many species including the rat [3, 4], mouse [7], pig [8], ferret [10], and human [2]. Mesenchymal cells of the testis interstitium embryologically originate either from the mesonephric tubules (invade the developing gonadal ridge and ultimately contribute to the developing testicular cords and testis interstitium) or loose connective tissue of the developing gonad originating from the embryonic mesoderm [12]. While some of the mesenchymal cells in the testis interstitium differentiate into fetal Leydig cells and many other cell types in the testis interstitium during gestation, others retain their undifferentiated characteristics and serve as precursor cells for the adult Leydig cells in the postnatal testis [12]. These undifferentiated mesenchymal cells are abundant in the postnatal testis interstitium at the time when the process begins. According to early studies, the postnatal porcine Leydig cell differentiation proceeds in a wavelike manner with the differentiation of fusiform cells into polygonal Leydig cells [8]. The process is said to begin with formation of smooth endoplasmic reticulum (SER) in one pole of the cell at first but becomes abundant with SER, mitocondria, and many other organelles with the progression of the process [8].

Precursor mesenchymal cells are seen at the peritubular area as well as in nonperitubular regions (can also be described as centrally positioned or randomly scattered) of the testis interstitium. Early reports [3, 13–15] support the view of peritubular mesenchymal cells as the primary precursor for adult Leydig cells. The only report that opposes this view is by Hardy et al. [16], and according to these investigators, the fusiform cells in the central interstitium are precursors to the adult Leydig cells instead. The basis for this conclusion is not apparent from the data presented in that report [16], because the criteria for identifying the precursor cells are not clear, e.g., precursor-progenitor cell markers were not used. Several recent studies unequivocally demonstrated peritubular mesenchymal cells as precursors to the adult Leydig cell population in the prepubertal rat testis at the onset of Leydig cell differentiation [17–19] (Fig. 2) by using 3ß-hydroxysteroid dehydrogenase (3ß-HSD; a universally accepted marker for all steroid-secreting cells) as well as two other steroidogenic enzymes, cytochrome P450 side chain cleavage (P450scc) and cytochrome P450 17{alpha}-hydroxylase (P450c17). In addition, peritubular mesenchymal cells have been identified as precursor cells for Leydig cells in the ethane dimethane sulfonate (EDS)-treated adult rat testis during regeneration of Leydig cells [20, 21]



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  		FIG. 2. Representative light micrographs (A–D) of progenitor cells (arrows) containing 3ß-HSD enzyme activity, differentiating toward early adult Leydig cells in postnatal rat testes later on Day 10. The elongated spindle-shaped configuration gradually transforms into a rounded shape while moving from the peritubular location. (Bouin-fixed paraffin-embedded tissue; bar = 20 µm.)

Progenitor Cells

The first step in the Leydig cell differentiation process is transformation of precursor cells into Leydig cell progenitors. Cellular or molecular mechanisms of the steps in progenitor cell formation have not yet been fully described. Leydig cell progenitors are morphologically indistinguishable from precursor cells, but they are definitely committed toward the Leydig cell lineage because they express steroidogenic enzymes (Fig. 2) [18, 22–24] and are capable of producing androgens [24–27]. It has been claimed that Leydig cell progenitors have LH receptors [26–29], and this has been confirmed in recent studies [17]. However, it is uncertain whether Leydig cell progenitors are responsive to LH at the beginning of their differentiation, because it is reported that these cells contain nonfunctional form of LH receptors (based on transcript length, not protein) consisting only of extracellular domain [30, 31], and only a small amount of LH binding to them can be demonstrated [26]. However, as there is no evidence to show that LH binds to the truncated LH receptor, an alternative explanation is that LH could be binding to a smaller number of full-length receptors.

Recent studies in the prepubertal rat testis demonstrated that the first detection of progenitor cells in the testis interstitium is later on Postnatal Day 10 (Fig. 2) [16]. In addition, this study revealed that when precursor cells differentiate into progenitor cells of the Leydig cell lineage, they simultaneously acquire 3ß-HSD, cytochrome P450scc and P450c17 [16]. More importantly, this study demonstrated that the progenitor cells acquire these steroidogenic enzymes prior to gaining LH receptors [16]. Latter findings suggest that the onset of precursor cell differentiation in the prepubertal rat testis is independent of LH, similar to what occurs in the fetal testes in rats [32] and mice [33]. It is evident that with the progression of the differentiation of the progenitor cells toward the next cell type (i.e., the newly formed adult Leydig cells), they undergo a change in shape, becoming rounder in configuration, and they begin to move from their peritubular position toward the central interstitium (Fig. 2). Mechanisms underlying these changes are still to be established.

Newly Formed Adult Leydig Cells

The next step in the process of Leydig cell differentiation is transformation of progenitor cells into morphologically identifiable adult Leydig cells (see Fig. 1). The most obvious difference between progenitors and the newly formed early adult Leydig cells is the change in cell shape from spindle-shaped to polygonal. In addition, this differentiation is accompanied by movement of the cell toward the central interstitium, away from its peritubular origin. Therefore, the newly formed adult Leydig cells are found also in the central interstitium. These newly formed adult Leydig cells are smaller than the immature [34] and mature adult Leydig cells [11, 34]. They could be differentially identified from the immature Leydig cells based on their characteristic morphology other than the cell size. The cytoplasmic content of the newly formed adult Leydig cells is sparse, and therefore, the nucleus appears rather large and prominent. Additionally, they contain little or no cytoplasmic lipid droplets in them (Fig. 3) [11, 35–37]. They stain intensively for steroidogenic enzymes (Fig. 4) [17–20, 22, 38, 39] as well as for LH receptor [40]. The newly formed adult Leydig cells do not contain 11ß-hydroxysteroid steroid dehydrogenase 1 (11ß-HSD1) enzyme immediately after they differentiate from progenitors but gain this activity gradually from Postnatal Day 21 onward [37] while they were differentiating into the immature Leydig cells. It appears that an early adult Leydig cell has a 40% of the testosterone secretory capacity of a mature Leydig cell and the greatest capacity for secreting androstenedione when compared to the immature and mature adult Leydig cells in the postnatal rat testis (Fig. 5). The results in Figure 5 were calculated by using the data published previously by Ariyaratne and Mendis-Handagama [34]. This cell type (i.e., the newly formed adult Leydig cells) in the Leydig cell lineage has been documented as early as 1959 [1] and in many other reports thereafter [3, 11, 17–19, 34, 37, 41]; however, it is missing in reviews on Leydig cell differentiation by Benton et al. [42] and Ge et al. [43], and the reasons are unclear.



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  		FIG. 3. A representative light micrograph of a newly formed adult Leydig cell at Postnatal Day 21 in the rat testis (used with permission of the publisher [35])



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  		FIG. 4. A representative light micrograph immunolabeld for 3ß-HSD enzyme in newly formed adult Leydig cells at Postnatal Day 21 in a rat testis. Note that the newly formed adult Leydig cells appear as rounder profiles and are found near the peritubular region as well as away from the tubules (used with permission of the publisher [18])



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  		FIG. 5. Luteinizing hormone-stimulated testosterone (A) and androstenedione (B) secretory capacity per adult type Leydig cell in rat testes during development (calculated from the results of Prince [35])

Immature Adult Leydig Cells

The size of the newly formed adult Leydig cells increases steadily with age [34] by acquiring more cytoplasm and becoming immature adult Leydig cells. Immature adult Leydig cells are quite distinct from newly formed adult Leydig cells and mature adult Leydig cells, respectively, because Leydig cell cytoplasm is laden with lipid droplets (Fig. 1) [34, 44]. The total number of lipid droplets as well as the size of the individual lipid droplets in the immature adult Leydig cells appear to be smaller than those of fetal Leydig cells [13, 23, 41]. Some investigators [13, 34] were able to distinguish fetal Leydig cells from the immature adult Leydig cells in the prepubertal rat testis using this criterion, although these differences were not detected by others [43]. Nevertheless, the number of fetal Leydig cells per testis is extremely small compared to the number of immature adult Leydig cells per testis [34]. The immature adult Leydig cells are the dominant cell type of the Leydig cell lineage from Postnatal Days 26–28 onward to about Days 45–50 in the rat testis and could be unequivocally identified and distinguished from the fetal population of Leydig cells because the fetal Leydig cells do not contain 11ß-HSD1 activity [34, 37, 45]. In contrast to the newly formed adult Leydig cells, the whole population of immature adult Leydig cells is positive for the 11ß-HSD1 enzyme, and this feature demonstrates that the immature adult Leydig cells are functionally closer to the mature Leydig cells than the newly formed adult Leydig cells.

In the rat, the activities of P450scc, 3ß-HSD, and P450c17 enzymes of the steroidogenic pathway increase readily in the immature adult Leydig cells from Postnatal Day 28 through Day 56 [22, 36, 38, 46]. However, the activity of 17ß-hydroxysteroid dehydrogenase (17ß-HSD, also known as 17-ketosteroid reductase [17KSR]) remains lower until Postnatal Day 36, and as a result, the ability of the immature adult Leydig cells to produce testosterone is considerably lower than the mature adult Leydig cells [47]. Another contrasting aspect of steroid metabolism in the immature adult Leydig cells compared to fetal Leydig cells or mature adult Leydig cells is the presence of high levels of testosterone-metabolizing enzymes such as 5{alpha}-reductase and 3{alpha}-hydroxysteroid dehydrogenase [46, 48]. Therefore, these cells produce mainly 5{alpha}-reduced androgens such as androstane-3,17-ß-diol (3-diol) and androstenediol instead of testosterone [47, 49–53].

Mature Leydig Cells

The final step in adult Leydig cell development is the transformation of immature adult Leydig cells into mature adult Leydig cells. This transition is characterized by a significant increase in the average cell size [34] and disappearance of cytoplasmic lipid droplets [11, 13, 44]. The capacity to secrete testosterone increases significantly in mature adult Leydig cells because they acquire more organelle components necessary for steroid production and enhanced responsiveness to circulatory LH. This is possibly due to the acquisition of higher numbers of LH receptors [26]. With the gradual increase in organelle volumes such as SER, these cells gain more steroidogenic enzyme activity, particularly 17ß-HSD that catalyzes the final step in testosterone biosynthesis [47]. A dramatic loss of testosterone-metabolizing enzymes occurs during this time as well [46, 48], which also contributes to the increased testicular testosterone-secreting capacity of the mature adult Leydig cells. With the accomplishment of this transition, puberty is reached.


   LEYDIG CELL NUMBERS
TOP
 ABSTRACT
 INTRODUCTION
 LEYDIG CELL LINEAGE
 LEYDIG CELL NUMBERS
REGULATION OF POSTNATAL...
 REFERENCES
 
Prepubertal and pubertal increases in Leydig cell number in the postnatal testis appear to be brought about by two mechanisms, differentiation of mesenchymal cells into Leydig cells and the mitotic division of newly formed Leydig cells that is reported to occur during a limited period [54, 55]. Although there is no doubt that both of these mechanisms are involved in establishing the adult Leydig cell population, the extent of the contribution by each source to this process is yet uncertain. Hardy et al. [16] proposed two distinct phases in the differentiation of Leydig cells in the postnatal rat testis. The first phase is between Postnatal Days 14 and 28, which is solely the transformation of mesenchymal cells to Leydig cells, whereas the second phase begins from Day 28, and Leydig cell numbers are increased as a result of Leydig cell division. Results of other investigations do not agree with the above conclusions because these investigators observed proliferation of adult Leydig cells much earlier than Day 28 postpartum [13, 39]. Recent observations of Ariyaratne et al. [18, 19] were in agreement with the latter investigators [13, 39], because they observed proliferation of progenitors as well as newly formed adult Leydig cells immediately after they were differentiated in the prepubertal rat testis. In addition, recent studies have shown that both Leydig and mesenchymal cell numbers per testis increase from birth to sexual maturity in the rat [34]. These findings differ from an earlier study [16] that reported an initial increase in mesenchymal cell numbers from birth to Day 28, but a decrease thereafter reaching a 50% value at Day 56 compared to Day 28. It is difficult to explain how this could occur when the testis is continuously increasing in volume during this growth period and how a 485-mm3 testis (at Day 28) could have twice the number of mesenchymal cells compared to a testis with a volume of 1225 mm3 (at Day 56). Another controversial issue is the existence of a precursor-product relationship between mesenchymal and Leydig cells proposed by Hardy et al. [16], where an increase in one Leydig cell per testis is accompanied by a net loss in one mesenchymal cell per testis during postnatal Leydig cell differentiation, based on cell percentages. Based on absolute cell numbers per testis, it is evident that at a given time from birth to 90 days of age in the rat, approximately two Leydig cells are generated when one mesenchymal cell is added to the testis interstitium [34] (Fig. 6). This relationship between mesenchymal and Leydig cell numbers has also been observed during Leydig cell differentiation in rats subjected to transient neonatal hypothyroidism (unpublished data). Therefore, the idea that an increase in each Leydig cell per testis is accompanied by a net loss of one mesenchymal cell during postnatal Leydig cell differentiation in the rat as suggested previously [16] is no longer valid. Recent cell quantification studies in EDS-treated adult rat testes have also shown that both the mesenchymal and Leydig cell number per testis increase during repopulation of the testis interstitium with Leydig cells following EDS treatment [21]. Clearly, the kinetics in Leydig cell differentiation is not a simple process that has clearcut phases of differentiation and mitosis, as well as one-to-one decreases and increases in cell numbers of mesenchymal and Leydig cells, respectively.



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  		FIG. 6. A) The absolute number of mesenchymal and Leydig cells in the postnatal rat testis increase with age with an approximate rate of 1:2, respectively. B) Interpretation of the results based on these cell numbers expressed as a percentage of the total interstitial cell numbers provides a misleading relationship (used with permission from the publisher [35])

The cellular mechanisms that result in the differentiation of the adult population of Leydig cells continue to function in the mature testis to maintain a constant number of Leydig cells. Although the mechanisms involved in maintaining this population in the adult testis are not yet well established, it is understood that the lost Leydig cells due to natural cell death are replaced with new Leydig cells. This process of Leydig cell renewal is demonstrable to some extent by removing existing Leydig cells by EDS in the rat testis [56].


   REGULATION OF POSTNATAL DIFFERENTIATION OF THE ADULT LEYDIG CELL POPULATION
TOP
 ABSTRACT
 INTRODUCTION
 LEYDIG CELL LINEAGE
 LEYDIG CELL NUMBERS
 REGULATION OF POSTNATAL...
REFERENCES
 
Circulatory hormones as well as locally produced growth factors are shown to have important effects on the differentiation and maturation of the adult Leydig cell population, and it appears that different factors regulate each stage of the Leydig cell lineage.

Luteinizing Hormone

Luteinizing hormone has been suggested in many instances [42, 57–59] as the key regulator of Leydig cell differentiation. Indirect evidence from several lines of investigations suggested that LH is not required at the onset of Leydig cell differentiation, although it is essential for the later stages in the Leydig cell lineage. These include the following observations. In immature rats, at the time of mesenchymal cell proliferation and differentiation into Leydig cell progenitors (i.e., Postnatal Days 10–11), the serum LH level is quite low, and it does not begin to rise until much later [60–62]. Continuous proliferation of the mesenchymal cells and the differentiation of these cells into some early form of cells in the Leydig cell lineage have been detected in the testicular interstitium of rats either after hypophysectomy or suppression of LH by testosterone implants [63, 64]. In transiently hypothyroid rats, Leydig cell precursors continue to proliferate during the hypothyroid period, and these precursors differentiate into adult Leydig cells when the animal becomes euthyroid [65] despite the permanently depressed plasma LH levels throughout the experimental period [66]. In the hypogonadal (hpg) mouse that has a congenital deficiency in GnRH and therefore a lack of exposure of the gonads to endogenous LH, Leydig cells are present in the testis interstitium, although few in number [67]. Without exogenous LH, these mice remain in a prepubertal, less undifferentiated state as evidenced by exhibiting very low levels of relaxin-like factor (RLF, also known as Ley-IL) [68], which is a product of the INSL3 gene and a marker for fully differentiated Leydig cells [56, 68]. Most recent studies have revealed that the adult population of Leydig cells differentiate in LH receptor knockout mice, although the numbers of these cells are considerably lower than those of control mice [69] (I.T. Huhtaniemi, personal communication). These observations are in agreement with the concept that the onset of mesenchymal cell differentiation is independent of LH, but all steps beyond this point are critically dependent on this hormone.

Other direct evidence to support the view that LH is not required for the onset of precursor cell differentiation into progenitor cells in the postnatal testis to begin the process of Leydig cell differentiation comes from two recent studies. The first study showed that Leydig progenitor cells in prepubertal rat testes gained steroidogenic enzymes prior to gaining LH receptors [17]. Second, daily s.c. injections of LH to rat pups from birth to weaning resulted in a delay in the differentiation of precursor cells to Leydig cell progenitor cells [19]. These findings strongly suggest that the onset of the process of adult Leydig cell differentiation is independent of LH. However, it is important to note that LH is required for the later stages in the lineage, i.e., proliferation of progenitors and their differentiation to newly formed adult Leydig cells, newly formed adult Leydig cells to undergo cell hypertrophy and differentiate into immature and mature adult Leydig cells, as well as for the mitosis of Leydig cells following their differentiation. The view on the LH requirement for Leydig cells to undergo cell hypertrophy is supported by the presence of smaller Leydig cells under conditions of LH deficiency, such as in following transient neonatal hypothyroidism [65], hypophysectomy [70], and testosterone-estradiol silastic implants [71]. Moreover, the importance of LH in Leydig cell hypertrophy and hyperplasia is evident by studies on LH treatment of rats [72] and hpg mice [73], and hCG treatment of rats [74].

Androgens

Evidence for the possible involvement of androgens produced by the fetal Leydig cells in the neonatal-prepubertal testis on adult Leydig cell differentiation comes from several experimental and naturally occurring conditions. Available data indicate that androgens are stimulatory to some steps of the Leydig cell lineage while inhibitory to others. Elimination of fetal Leydig cells using EDS during the first week of birth advances and stimulates adult Leydig cell differentiation [75], while stimulation of fetal Leydig cell proliferation by administration of either hCG or LH results in inhibition [76] or delay [20], respectively, of adult Leydig cell differentiation, indicating the possible inhibitory effects of testosterone on early stages of adult Leydig cell development in the rat. Androgen treatment also could not be shown to produce any effects on Leydig cells in hpg mice [67].

The effect of androgens on Leydig cell differentiation appears to be direct because all cellular stages in this pathway express androgen receptors [26, 43, 77, 78]. It appears that androgens are required for the maturational acquisition of steroidogenic enzymes in later steps of the Leydig cell lineage. Expression of androgen receptor mRNA and protein levels is highest in immature adult Leydig cells, and this finding suggests that the conversion of these cells into mature adult Leydig cells may be the most androgen-sensitive step in Leydig cell differentiation [79]. It has also been reported that the differentiation of Leydig cell progenitors into immature adult Leydig cells in vitro depends on the presence of both LH and dihydrotestosterone [24]. Moreover, in LHRH-antagonist-treated immature rats, the normal morphology and function of Leydig cells could be restored only after androgens were administered to these animals [78, 80]. In testicular feminized (Tfm) mice that have a nonfunctional mutation of the androgen receptor leading to androgen insensitivity, the main defect is not the lack of progenitor cells in the testicular interstitium but failure of the progenitor cells to differentiate into the adult Leydig cells and inability to acquire some steroidogenic enzymes, such as P450c17 and 17ß-HSD [81–85]. In Tfm mice, androgen production is severely reduced [82–84], despite the high plasma LH levels and normal number of hyperplastic Leydig cells. The main lesion detected in the steroidogenic pathway of these Tfm mice is the very low level of P450c17 activity [83, 85, 86] and 17ß-HSD [83], indicating the necessity of androgens for the expression of these enzymes.

Estrogens

The effects of estrogens on Leydig cell differentiation appear to be dependent on the stage of Leydig cell lineage. The absence of mature Leydig cells in testes of 60-day-old rats who received a single injection of estradiol at 5 days of age [87] suggests that Leydig cell development is sensitive to estrogens. Further evidence for the estrogen influence on Leydig cell differentiation comes from studies of Abney and Myers [88], who demonstrated that regeneration of Leydig cells in the mature rat testis after EDS treatment was prevented by daily injections of estradiol given during the period from Day 5 to Day 16 after EDS treatment, but Leydig cell regeneration was not prevented if the estradiol injections were restricted to Days 0–5 following EDS. These investigators suggested the occurrence of important developmental process that is necessary for Leydig cell regeneration between Days 5 and 16 post-EDS, and estrogen is inhibitory to this event. It is known that during early postnatal life in the rat (Days 5–10), Sertoli cells are the primary source of testicular estrogens [89, 90]. Testicular estrogen levels decline during the period of Postnatal Days 10–21 [91], which correspond with the onset of precursor cell differentiation into progenitors and then to the early adult Leydig cells [11, 19, 30]. It is also important to note that testicular estrogen production increases again [92, 93] with the emergence of adult Leydig cells [77]. In other studies, 3H-thymidine incorporation by the testicular interstitial cells from mature rats both in vivo [94] and in vitro [95, 96] was inhibited by estrogen in the presence and absence of hCG. Therefore, the estrogen produced by the Leydig cells at later stages could be viewed as an important necessary factor in inhibiting the differentiation of precursor cells to regulate further production of Leydig cells [92, 93].

Follicle-Stimulating Hormone

Early studies suggested that FSH affects Leydig cell development, acting via Sertoli cells [93]. The first clue for this concept was derived from the studies using hypophysectomized immature rats. In these rats, treatment with FSH, but not with LH, was able to increase the number of testicular LH receptors. Moreover, FSH was observed to enhance the capacity of the testis to secrete testosterone in response to LH as well as testicular size in these rats [97–101]. Using hypophysectomized immature rats, it was also shown that FSH treatment increased both the Leydig cell number and the size of individual Leydig cells by promoting the differentiation of adult-type Leydig cells [102, 103] and stimulated the functional maturation of these cells by inducing steroidogenic enzymes [102]. However, one important aspect of FSH function on Leydig cells demonstrated in these studies was an obligatory need for the presence of small amounts of LH for FSH action [104], because pure FSH was without effect. In contrast to these early studies that used impure preparations of FSH, studies performed much later with pure preparations of FSH revealed that FSH is not required for the development of Leydig cells. These studies were performed in immature hypophysectomized rats [105] and adult hypophysectomized rats treated with EDS [106].

Anti-Müllerian Hormone

Anti-Müllerian hormone (AMH), also called the Müllerian-inhibiting substance (MIS) or Müllerian-inhibiting factor (MIF), is a member of the transforming growth factor ß family of cytokines that includes transforming growth factor ß, activins, inhibins, and the bone morphogenetic proteins. However, in contrast to the complexity of other TGFß gene family signaling pathways, AMH is the only ligand of the AMH type II receptor [107]. Anti-Müllerian hormone induces regression of the Müllerian ducts in the developing male fetus [108, 109] and it is a product of the immature Sertoli cells. Immunocytochemical studies on rat testes have revealed that labeling of Sertoli cells for AMH decreases gradually after the third postnatal day, dramatically on the fifth postnatal day, and is present at a very low level about the 20th postnatal day [110]. Although the factors that regulate AMH production by Sertoli cells in the postnatal testis are still to be established, the postnatal decrease in AMH production by Sertoli cells of immature rats is said to be regulated at least in part by triiodothyronine (T3) and FSH [111]. In addition, it has also been shown that AMH immunolabeling and AMH mRNA could be considerably decreased with FSH treatment; by contrast, LH treatment produced no effects [110]. It has been documented that changes in AMH protein content occur in the absence or with minimal changes in AMH mRNA levels [112], suggesting that measurement of AMH mRNA is not an accurate index of AMH production-expression. According to some reports, AMH is present in Sertoli cells at sexual maturity [113, 114], but it is also documented that AMH is absent in Sertoli cells in adult testes that has normal spermatogenesis [115], however, it is present when there is spermatogenic arrest [115].

Anti-Müllerian hormone is known to have roles in Leydig cell steroidogenesis [116], maturation of gonocytes to type A spermatogonia in the neonatal mouse testes [109], and in spermatogenesis in the adult rat testis [117]. A possible involvement of AMH in Leydig cell development was revealed in studies of transgenic mice. Male transgenic mice that chronically overexpress human AMH (hAMH) under the control of the mouse metallothionein-1 promoter (MT-hAMH mice) have undescended testes, are incompletely masculinized externally, and rapidly become infertile [114, 115], suggesting a defect in androgen biosynthesis. Subsequent studies have shown that male transgenic mice that ectopically express AMH have depressed levels of circulating testosterone, suggesting that excess hAMH can alter Leydig cell function [118].The tissue-specific AMH-induced mutant phenotypes of transgenic mice with high levels of circulating AMH suggest that the limiting factor in this signal transduction pathway is the AMH type II receptor, which is now known to be localized to the Leydig cells [119]. Additionally, studies of Racine et al. [119] have suggested that overexpression of AMH in male transgenic mice blocks the differentiation of Leydig cell precursors in the postnatal testis to establish the adult Leydig cell population.

Growth Factors

Insulin-like growth factor-I (IGF-I) The possible role of IGF-I on testicular function has been suggested based on experimental and clinical data [120, 121]. The stimulatory effects of IGF-I on Leydig cell differentiation were first observed in Snell dwarf mice that are characterized by low circulatory growth hormone and testosterone levels, delayed puberty, and poor response to exogenous gonadotropins [122]. Administration of IGF-I to these animals increased testicular LH receptors and LH-stimulated biosynthesis of androgens from immature type Leydig cells found in them [123]. In vitro studies using isolated Leydig cells from prepubertal rats have demonstrated that IGF-I stimulates proliferation of immature Leydig cells. Pretreatment of these cells with LH augmented this mitogenic effect [124–127]. Moreover, immature Leydig cells [128, 129] but not Leydig progenitor cells [130] respond to IGF-I by enhanced expression of steroidogenic enzymes and steroid production, suggesting that the effect of this growth factor is associated with promotion of the maturation of immature adult Leydig cells into mature adult Leydig cells. Recent studies using knockout mice for the IGF-I gene further strengthened this hypothesis [43]. Leydig cells in adult IGF-I null mutants are immature, contain little P450c17 and 17-KSR:17ß-HSD enzyme activities, and secrete mainly progesterone and androstenedione [43].

Transforming growth factors {alpha} and ß (TGF{alpha} and TGFß) Using immunocytochemical techniques TGF{alpha} activity has been detected in the prepubertal rat testicular interstitium. At 21 days of age, approximately 50% of Leydig cells stained positively for this growth factor [131], and at puberty all Leydig cells stained strongly for TGF{alpha}. The presence of the receptors for TGF{alpha} in rat Leydig cells has also been demonstrated by using similar methods [132]. However, based on the observations that TGF{alpha} binds to mesenchymal precursors rather than to mature adult Leydig cells, Moore and Morris [126, 127] suggested that the paracrine effects of TGF{alpha} may be mediated through interstitial mesenchymal cells. In vitro experiments using isolated Leydig cells from Day 21 rats by Khan et al. [124, 125] showed that TGF{alpha} stimulates the incorporation of [3H]thymidine to these cells, and the mitogenic activity of this growth factor is dependent on cell density and the presence or absence of LH.

Immunolocalization studies of TGFß in the rat testis have shown that all Leydig cells are positive for TGFß antigen at 7 days of age [133] only fetal Leydig cells are present at this age). Thereafter, the percentage of positively labeled cells declined gradually, and by 21 days of age only 50% of the Leydig cells were positive for TGFß [133]. However, it is not clear from these studies whether these labeled cells are fetal or adult types. The proportion of cells immunolabeled for TGFß was further reduced with advancing age, and by Day 35 postpartum no specific labeling was detected in the interstitial tissue [133]. According to studies by Khan and coworkers [124, 125] TGFß alone can stimulate [3H]thymidine incorporation into Leydig cell DNA, and this response is enhanced after pretreatment of these cells with LH. However, relative to TGF{alpha}, the mitogenic activity of TGFß is low. Based on these and other observations, Teerds [131] postulated that the effectiveness of LH in stimulating Leydig cell proliferation is dependent on the relative amounts of TGF{alpha}, TGFß, IGF-I, and interleukin (IL)-1ß present. Luteinizing hormone requires TGF{alpha} together with IGF-I or IL-1ß to stimulate proliferation of Leydig cells, while the action of these factors is abolished by the presence of TGFß.

Platelet-derived growth factor-A (PDGF-A) Platelet-derived growth factor-A is known as a key regulator of connective tissue cells in embryogenesis and pathogenesis [134]. It is also a mitogen and a chemotactic factor for mesenchymal cells [135] and is present in Leydig cells [136, 137]. Although it is suggested that PDGF-A is an essential factor for differentiation of adult Leydig cells [138], little is known about the mechanisms underlying its participation in Leydig cell differentiation.

Macrophage-Derived Factors and Cytokines

Little is known about the effects of cytokines on Leydig cell development in comparison to their known influences on the steroidogenic function of these cells [120, 139]. It has been shown that similar to the other circulating or residential macrophages in other organs, testicular macrophages also secrete a number of cytokines [140, 141], some of which are mitogenic to Leydig cells [124, 125]. Retardation or absence of Leydig cell development has been observed in experimental models with impaired macrophage function [142, 143] When macrophages are depleted from the neonatal testis by treatment with dichloromethylene diphosphonate-containing liposomes, development of adult Leydig cells failed completely in treated animals [142]. Moreover, in osteoporotic mice whose testes contain only a few macrophages due to the lack of colony-stimulating factor-1, which is essential for the proliferation and survival of these cells, the Leydig cells are quite abnormal in appearance and functionally retarded [139].

Thyroid Hormone

The effect of thyroid hormone on proliferation and maturation of Sertoli cells in the postnatal testis has been the subject of many studies [144–152]. However, little has been known about the effects of thyroid hormone on the development of testicular interstitial cell types for some time. In transient neonatal hypothyroid rats that were made hypothyroid for the first 25 days after birth and allowed to become euthyroid thereafter, testes contained twice the number of Leydig cells per testis compared to similar age controls at Day 135 [65]. These studies also showed that the average volume of a Leydig cell in these hypothyroid rats was significantly smaller than in control rats [65]. However, serum testosterone levels and LH-stimulated testicular steroidogenic capacity (testosterone, androstenedione, 17-hydroxyprogesterone, and progesterone) in vitro were not different between the two groups [65]. The mechanism by which neonatally hypothyroid rats acquire more Leydig cells was difficult to explain at that time. However, one speculation was that transient neonatal hypothyroidism may have resulted in more Leydig cell precursors [65]. It was shown later that this view may be true, because increased numbers of precursor cells are generated in the prepubertal testes during the hypothyroid period [37] due to the absence of their differentiation under a hypothyroid status as discussed in the next paragraph [37, 39]. Therefore, it is logical to suggest that when the animal becomes euthyroid after the withdrawal of propylthiourea treatment, these excess precursor cells differentiate to produce increased numbers of Leydig cells. By contrast, it is reported elsewhere [153] that increased proliferation of adult Leydig cells from Day 8 through Day 50 postpartum rather than increased proliferation of their mesenchymal precursors is the principal mechanism responsible for the increased numbers of Leydig cells in rats subjected to transient neonatal hypothyroidism. Evidence to reject this conclusion comes from two strong lines of evidence. First, it is now established that adult Leydig cells do not differentiate in the postnatal rat testis prior to Postnatal Day 10 [11, 18]. Second, it has been repeatedly shown that adult Leydig cell differentiation does not take place in the prepubertal rat testis under a hypothyroid state [19, 37, 39]. The only Leydig cell type present in the postnatal testis interstitium at this time is the fetal Leydig cell. The Leydig cell in mitosis presented in that report [153] has all the characteristics of a fetal Leydig cell, although it has been presented as an immature adult Leydig cell to support that view. Moreover, Teerds et al. [39] could not observe a difference in the labeling index for Leydig cells in hypothyroid and control animals at any age during the neonatal-prepubertal period [39], although Hardy et al. [153] showed that Leydig cell proliferation is present in hypothyroid rats but absent in control rats at these early ages. Nevertheless, the latter finding contradicts a previous report by the same investigators [16] where Leydig cell proliferation is reported in control rats during the neonatal-prepubertal period. It is difficult to comprehend these discrepancies because the two reports come from the same investigators.

The regulatory effects of thyroid hormone on Leydig cell differentiation were revealed more recently. It is known that Leydig cell differentiation is arrested in the absence of thyroid hormone in the prepubertal [19, 37, 39] as well as in the EDS-treated adult rat testis [21]. It has also been demonstrated that hyperthyroidism causes accelerated Leydig cell differentiation [18, 39]. As revealed by immunocytochemistry for 3ß-HSD, it is evident that not only Leydig cells, but Leydig progenitor cells are also absent in hypothyroid rats [19]. These findings suggest that the differentiation of mesenchymal cells into Leydig progenitor cells requires thyroid hormone. This suggestion is further supported by the observation that premature recruitment of mesenchymal cells in greater numbers into the Leydig cell lineage than in control rats occurs in the prepubertal rat testis under hyperthyroid conditions [18, 19, 39]. These results imply that thyroid hormone causes proliferation of mesenchymal cell precursors and acceleration of their differentiation into Leydig cell progenitors in addition to its effect of enhanced proliferation of progenitors and newly formed Leydig cells [18, 19].

The EDS-treated adult rat model has been used extensively to study Leydig cell differentiation in the postnatal rat [56]. Recently, the EDS model was employed to investigate the effects of hyperthyroidism and hypothyroidism on Leydig cell development in the adult rat [21]. The results revealed that hypothyroidism prevented Leydig cell regeneration after EDS administration, and hyperthyroidism resulted in an early onset of mesenchymal cell differentiation together with increased numbers of Leydig cells as compared with controls at Day 21 following the treatment. This is reminiscent of what is observed with thyroid hormone regulation of Leydig cell differentiation in the prepubertal rat testis [18, 19]. Whether the effect of thyroid hormone on the process of Leydig cell differentiation is direct or indirect has yet to be determined. It is possible that thyroid hormone directly initiates the mesenchymal cells to differentiate into Leydig cell progenitors or it may act via inducing Sertoli cell maturation and thereby inhibiting AMH production as reviewed above.

Light

Seasonally breeding mammalian males show profound testicular atrophy during the nonbreeding season [154]. Testes of hamsters exposed to short day lengths have reduced capacity to secrete testosterone [155]. It has also been reported that Leydig cells in hamsters exposed to short day lengths (e.g., 6L and 18D) undergo cell atrophy [156, 157] and hypoplasia [156–158]. Stereological studies have revealed a decrease in volumes of Leydig cell organelles, especially the ones that are associated with steroidogenesis [157]. However, inducing recrudescence by switching the short days to long days (14L and 10D) results in restoration of the Leydig cell number and size [156, 157]. As light is an important factor in maintaining the differentiated status of Leydig cells in such species, it may possibly have a regulatory role in the process of their postnatal differentiation of Leydig cells, which warrants further investigation.

Factors regulating adult Leydig cell differentiation in the postnatal testis are summarized in Figure 7.



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  		FIG. 7. Schematic representation summarizing the regulation of adult Leydig cells differentiation in the postnatal testis. ALC, Adult Leydig cells; TH, thyroid hormone; +, stimulation; -, inhibition


   ACKNOWLEDGMENTS
 
The authors thank Phil Snow and Tommy Jordan in the Photography Laboratory of the College of Veterinary Medicine at The University of Tennessee, Knoxville, for their assistance in preparing the color photographic plates.


   FOOTNOTES
 
First decision: 8 November 2000.

1 Supported by grants from UT-COE R180101-08 and Professional Development Awards Program. Back

2 Correspondence. FAX: 865 974 2215; mendisc{at}utk.edu Back

Accepted: March 21, 2001.

Received: October 30, 2000.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 LEYDIG CELL LINEAGE
 LEYDIG CELL NUMBERS
 REGULATION OF POSTNATAL...
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