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Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Address all correspondence and requests for reprints to: William H. Walker, S333 Biomedical Science Tower, Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. E-mail: walkerw{at}pitt.edu.
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Introduction |
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 Top Introduction Note Added in ProofReferences |
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Previously, the mechanisms responsible for halting the proliferation of Sertoli cells and initiating their differentiation were not clear. In rodents, exit from the cell cycle does not appear to be due to limited proliferative signals because serum levels of the major hormone that supports Sertoli cell proliferation, FSH, rise continually during early postnatal development (9). The expression of FSH receptors on Sertoli cells also increases during this period (10). Furthermore, supraphysiological levels of FSH are capable of increasing the frequency of cell division and, thus, the final number of Sertoli cells, but such treatments do prolong the proliferation period (11). Based on these findings, studies have focused on the actions of negative factors that may be responsible for Sertoli cell exit from the cell cycle.
In this issue of Endocrinology, the studies presented by Holsberger et al. (12) and Buzzard et al. (13) provide evidence for thyroid hormone being an initial signal required to limit Sertoli cell proliferation. Also, testosterone and retinoic acid are shown to inhibit the proliferation of immature Sertoli cells. More importantly, these studies are the first to address the molecular mechanisms that cause Sertoli cells to exit the cell cycle and initiate differentiation. Specifically, the cell cycle inhibitory proteins p27Kip1 and p21Cip1 are identified as being induced in response to stimulation by thyroid hormone, testosterone, or retinoic acid (13). p27Kip1 and p21Cip1 are expressed at higher levels in many cell types undergoing exit from the cell cycle and terminal differentiation (reviewed in Ref.14). These proteins bind to and inhibit the activity of the cyclin-dependent kinases Cdk2 and Cdk4 that are required for cells to pass from the G1 to S phase of the cell cycle (15).
The studies by Holsberger et al. (12) link thyroid hormone stimulation to the induction of cell cycle inhibitors, as Sertoli cell p27Kip1 protein levels are higher in hyperthyroid compared with euthyroid mice. Furthermore, p27Kip1 levels were found to increase in euthyroid mice from d 5 postpartum, when Sertoli cells are dividing, to 16 d after birth when Sertoli cells are exiting the cell cycle. Buzzard et al. (13), using cultured Sertoli cells isolated from 6-d-old rats, demonstrate that, compared with control treatments, thyroid hormone stimulation slows Sertoli cell proliferation by greater than 50 and 80% after 4 and 8 d, respectively. These studies also revealed that retinoic acid or testosterone similarly inhibit Sertoli cell proliferation. Interestingly, various combinations of thyroid hormone, retinoic acid, and testosterone did not result in additive effects. The lack of synergism displayed in culture conditions raises the possibility that the hormones act through the same pathway to signal the end of Sertoli cell expansion, although further studies will be required to rule out the possibility that the hormones might synergize to slow Sertoli proliferation in vivo.
Additional data presented by Buzzard et al. (13) suggest that thyroid hormone, testosterone, and retinoic acid can impact regulation of the cell cycle by a similar mechanism, that being the induction of p21Cip1 and p27Kip1. Over a 4-d period, thyroid hormone stimulation elevated p21Cip1 and p27Kip1 levels in cultured Sertoli cells by 75 and 95%, respectively. This induction of p21Cip1 and p27Kip1 is similar to that observed in differentiating epiphyseal chondrocytes over 7 d of thyroid hormone stimulation (16). Testosterone or retinoic acid also induce the expression of p21Cip1 and p27Kip1 as well or better than thyroid hormone. Specifically, the levels of the cell cycle inhibitors are increased 80–475% by these hormones. With the caveat that comparisons to untreated cells over the 4-d study are not provided, these data suggest that thyroid hormone, testosterone, and retinoic acid are each capable of inducing cell cycle inhibitors in Sertoli cells and that this mechanism may be used to initiate terminal differentiation of the Sertoli cell. However, the question of which factor ultimately triggers Sertoli cell exit from the cell cycle in vivo still remains to be determined.
Previously, there has been little evidence that testosterone or retinoic acid was capable of directly regulating Sertoli cell proliferation. In contrast, there is support for thyroid hormone as the signal for Sertoli cells to exit the cell cycle, including the findings that hyperthyroidism or administration of thyroid hormone to Sertoli cells in culture causes an early halt to their proliferation (5, 17). In addition, reduction of thyroid hormone levels by chemically induced hypothyroidism increases the percentage of dividing Sertoli cells in rats 10 d and older by 4-fold and extends the proliferation period of Sertoli cells to 25–30 d (18, 19). Interestingly, the increase in Sertoli cell number in hypothyroid models occurs despite FSH levels that are reduced 30–80% (18, 20). It would be interesting to study the hypothyroid model further to determine whether the levels of p21Cip1 and p27Kip1 in Sertoli cells are elevated after extension of the proliferative period to 25–30 d. Also, the question remains as to what finally causes Sertoli cells to stop dividing in this model—the lower levels of FSH or signals due to testosterone, retinoic acid, or some other factor.
The opportunity for thyroid hormone action on Sertoli cells appears to correspond to the period in which the rate of cell proliferation is decreasing. Serum levels of thyroid hormone increase from just above the level of detection on d 5 to peak on d 15 (9), whereas thyroid receptor levels in Sertoli cells decrease from d 5 until they are barely detectable on d 20 (21, 22, 23). Treatment of rats and mice with the reversible goitrogen 6-propyl-2-thiouracil (PTU) extends the period of Sertoli cell proliferation and increases final Sertoli cell numbers but is only effective if administration occurs during the early neonatal period when Sertoli cells divide rapidly (24). Together, these observations suggest that the effects of thyroid hormone are manifest between 5 and 15 d after birth.
Although progress now has been made toward understanding the molecular mechanisms that control Sertoli cell proliferation and differentiation, the pathways used by thyroid hormone, testosterone, and retinoic acid to elevate p21Cip1 and p27Kip1 expression in Sertoli cells remain to be identified. In other cell types, p21Cip1 is regulated at the level of transcription, whereas p27Kip1 appears to be regulated by posttranslational mechanisms that alter the half-life of the protein (25, 26). A next step would be to determine whether the cell cycle inhibitors are regulated by the same mechanisms in Sertoli cells. Because thyroid hormone, testosterone, and retinoic all act through steroid hormone receptors, it is possible that they may alter p21Cip1 and p27Kip1 expression by similar mechanisms. If true, this would explain the lack of synergism seen when inhibiting Sertoli cell proliferation. It would also be prudent to investigate thyroid hormone, testosterone, and retinoic acid effects on the expression of other cell cycle inhibitor proteins such as those of the Ink4 family (p16Ink4a, p15Ink4b, p18Ink4c, and p19Ink4d) that inhibit cyclin D-dependent Cdks.
In an attempt to identify genes associated with Sertoli cell differentiation that are altered by thyroid hormone, testosterone, or retinoic acid in culture, Buzzard et al. (13) assayed the expression patterns of five candidate genes after 4 d of treatment. Unexpectedly, thyroid hormone treatment alone did not significantly alter the expression levels for any of the selected genes. Furthermore, none of the hormones independently or in combination resulted in increases or decreases in gene expression of greater than 2-fold with the exception of retinoic acid-induced decreases in the expression of one gene (Dmrt) that is normally up-regulated and another (GATA-4) that is down-regulated during Sertoli cell differentiation. It is possible that more robust charges in gene expression might be evident with longer or shorter periods of hormonal stimulation, or that the expression of other differentiation-associated genes may be altered. However, the available data are consistent with the idea that the hormone-induced halting of Sertoli cell proliferation does not of itself drive the cultured cells to differentiate within the assay period of 4 d. Additional time may be needed, or other factors that are normally present in vivo may be required to initiate Sertoli cell differentiation.
Unfortunately, applying the lessons learned from Buzzard et al. (13) and Holsberger et al. (12) to Sertoli cells in higher primates, including men, is not straightforward. Unlike rodents in which the Sertoli cell proliferation period extends only from fetal development through the first 3 wk of life, most Sertoli cell expansion occurs over two distinct periods in rhesus monkeys and men. The first expansion in higher primates is similar to that of rodents in that Sertoli cell proliferation initiates during fetal development and continues for a defined time after birth. During the period from birth to 4–5 months of age, Sertoli cell numbers in rhesus monkeys are increased 4-fold, followed by a slower rate of proliferation illustrated by the 2-fold increase that was observed over the 10–12 months between infancy and the midpoint to puberty (27). Unique to higher primates is a second stage of Sertoli cell expansion during puberty when spermatogenesis is initiated (28, 29). In rhesus monkeys, a further 6-fold increase in Sertoli cells occurs during puberty before proliferation is halted (27). Similarly, in men there is also a marked increase of Sertoli cells during infancy and another 2-fold increase during puberty (28).
Higher primates may also differ from rodents in regard to the regulation of Sertoli cell proliferation by FSH and testosterone. The proliferation of Sertoli cells in higher primates during infancy and puberty corresponds to periods during which levels of FSH and testosterone are elevated (reviewed in Ref.2). In contrast to the antiproliferative effects of testosterone on Sertoli cells isolated by rats that was observed by Buzzard et al. (13), the elevation of testosterone levels in prepubertal rhesus monkeys increases the number of Sertoli cells (30, 31). Therefore, in higher primates, testosterone may not limit Sertoli cell expansion or the hormone may act only at specific developmental stages to repress proliferation. It is possible that, in higher primates, the relative strength of the proliferation signal from FSH vs. the antiproliferative signals due to thyroid hormone, retinoic acid, and perhaps testosterone determines whether Sertoli cells are in the cell cycle. FSH induces the expression of the cell cycle initiator cyclin D that counteracts the actions of p21Cip1 and p27Kip1 (32). In rhesus monkeys, the levels of testosterone as well as the elevated FSH concentrations that likely support Sertoli cell proliferation during infancy suddenly decline to barely detectable levels 4–5 months after birth. This situation may cause cyclin D expression in Sertoli cells to fall below the levels necessary to trigger cell division. Induction of p21Cip1 and p27Kip1 by antiproliferative hormones may or may not be required to limit Sertoli cell proliferation in this scenario. Clinical data from hypothyroid boys does not help to resolve this issue, because although testes were enlarged in 75% of the cases (presumably due to increased numbers of Sertoli cells supporting additional germ cells), FSH levels were also elevated in all cases (reviewed in Ref.33). In contrast, the situation during primate puberty is more similar to that of the 10- to 21-d-old rodent because FSH levels remain elevated. Thus, assuming that there is no reduction in sensitivity to FSH during puberty in higher primates, increased stimulation from thyroid hormone, testosterone, retinoic acid, or another factor would be predicted to be required for the induction of p21Cip1 and p27Kip1 and the halting of Sertoli cell proliferation as has been shown in the rodent model.
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Note Added in Proof |
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TopIntroductionNote Added in ProofReferences |
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Received June 18, 2003.
Accepted for publication June 24, 2003.
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References |
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TopIntroductionNote Added in ProofReferences |
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