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The Pediatric Endocrine Unit (M.M.L., C.C.S.), Pediatric Surgical Research Laboratory (P.T.M., P.K.D., D.T.M.) and Department of Pathology (F.I.P.), Massachusetts General Hospital, Boston, Massachusetts 02114; and The Population Council and The Rockefeller University (C.M.S., M.P.H.), New York, New York 10021
Address all correspondence and requests for reprints to: Mary M. Lee, M.D., Pediatric Endocrine Unit, Bartlett Hall Extension 410, 55 Fruit Street, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: mlee{at}helix.mgh.harvard.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leydig cells are believed to arise as two discrete populations. Fetal Leydig cells are discernable soon after testicular determination and remain steroidogenically active until birth (15). The majority of these cells are believed to dedifferentiate or undergo apoptotic cell death after birth (16, 17, 18). The few fetal Leydig cells that persist postnatally do not divide and have a negligible contribution to steroidogenesis. Postnatal Leydig cells are first recognizable in the prepubertal testis as progenitor Leydig cells (PLCs) that arise from undifferentiated mesenchymal stem cells and acquire steroidogenic capacity (19, 20). PLCs, which account for the majority of the Leydig cells in the prepubertal rat, undergo active cell division to establish the adult Leydig cell population (21, 22). During early sexual maturation, these cells differentiate into pubertal immature Leydig cells (ILCs) that are characterized by a much lower proliferative rate and higher steroidogenic capacity. By late puberty, the quiescent, highly steroidogenic adult Leydig cells that are characteristic of the sexually mature testis comprise the majority of the Leydig cell population.
Both MIS and its type II receptor are expressed in the peripubertal and early pubertal testis; thereafter, MIS receptor messenger RNA (mRNA) transcripts remain abundant (2, 4), whereas MIS expression progressively declines except for a transient modest rise peripubertally (5, 6, 23). The persistent expression of MIS and its receptor in the postnatal testis raised the possibility of additional roles for this hormone in male reproductive development. Initial studies localized the MIS receptor to Sertoli cells in the seminiferous tubule compartment of the testis (3, 4, 7), suggesting an indirect action of MIS upon the Leydig cells. These studies, however, focused on fetal and young postnatal testis, rather than sexually maturing testis. More recently, MIS receptor mRNA has been detected by RT-PCR in adult murine Leydig cells (24), and MIS has been shown to decrease net testosterone production in both fetal and adult primary Leydig cells (24, 25). We hypothesized that MIS acts directly upon Leydig cells via receptor mediated events to regulate their proliferation and steroidogenesis during testicular development. We speculated that this function of MIS occurs during the differentiation of Leydig cells from an undifferentiated stem cell to a pubertal phenotype with attainment of steroidogenic capacity. To test the hypothesis that MIS has direct actions in maturing Leydig cells, we developed a cell culture model using highly purified Leydig cells at different maturational ages. We then examined cell specific mRNA expression of the MIS type II receptor in the testis and characterized the binding and function of MIS in PLCs (21 day) and ILCs (35 day).
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Materials and Methods |
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Cell isolation and culture
PLCs and ILCs were isolated from testes collected at 21 and 35
days after birth, respectively, by Percoll density gradient
centrifugation as described previously (26, 27). Previous studies
showed that the majority of cells isolated at these two ages are
representative of the PLC and ILC maturational stages (19, 21). Sertoli
cell-enriched cultures were prepared at the same ages using the
seminiferous tubule fraction of cells collected during an early step of
the Leydig cell isolation protocol. Sertoli cells were isolated by
glycine treatment followed by sequential collagenase/hyaluronidase
digestion and filtration steps as described previously (28).
Cellular homogeneity and absence of germ cells were used as initial criteria for purity of the primary Leydig cell preparations, which was further confirmed by examination of Leydig cell specific 3ß-hydroxy-steroid dehydrogenase (3ßHSD) expression (29). The purity of the cultures was also assessed by Northern analysis of cell-type specific genes. Androgen binding protein (ABP) (gift of Dr. David Joseph, Biotechnology Development Institute at the University of Florida, Alachua, FL) (30), MIS (31), and the FSH receptor (gift of Dr. Michael Griswold, Washington State University, Pullman, WA) (32) were selected as genes specific to Sertoli cells, and the LH receptor (gift of Dr. Deborah Segaloff, University of Iowa College of Medicine, Iowa City, IA) (33) as a Leydig cell-specific gene.
Primary Leydig cells were cultured in serum-free DMEM/F12 (1:1) media supplemented with 15 mM HEPES, 26 mM sodium bicarbonate, 0.1% BSA, 100 mg/ml bovine lipoprotein (Sigma Chemical Co.), and 12 µg/ml gentamycin, in a reduced oxygen incubator (5%Co2/5% O2) (Nuaire TS Autoflow Co2/O2 incubator) (26, 27). The highly purified PLCs and ILCs were seeded at 2 x 106 cells/well in 6-well plates or 1 x 106 cells/well in 12-well plates and cultured overnight before addition of hormones. The cultures were treated with buffer or recombinant human MIS (3.5 to 172 nM) for 24–48 h, in the presence and absence of low doses of LH (1 ng/ml) (NIDDK-oLH-26, National Hormone and Pituitary Program).
Recombinant human MIS was purified by immunoaffinity chromatography from conditioned media of CHO cells stably transfected with a full-length genomic clone (34, 35). Because the MIS gene is conserved across species, it retains cross-species bioactivity (31, 36). The immunoaffinity-purified MIS was quantitated by ELISA and tested in a rat urogenital ridge organ culture assay for its bioactivity (37, 38). Recombinant human MIS, at doses of 0–5 µg, causes zero to complete regression in this rat bioassay (35).
The Leydig cells were labeled with 1 µCi/well [3H]thymidine (DuPont NEN, Boston, MA) during the last 4 h of hormonal treatment. After washing with Dulbecco’s PBS, the cells were harvested by gentle scraping and lysed in 0.5 ml hyamine hydroxide (ICN Radiochemicals, Irvine, CA), then counted in a liquid scintillation counter.
For individual experiments, mean thymidine incorporation for each condition was calculated from the triplicate wells. The results for each MIS dose were compared with that of buffer-treated controls and expressed as thymidine incorporation as a percent of mean uptake in the control wells. The results were then pooled as the mean of four to seven experiments for each experimental condition. The data were normalized and analyzed using one-way ANOVA to determine the overall effect of MIS treatment on thymidine incorporation. Tukey’s test was then used to evaluate all pair-wise comparisons, with unequal n’s adjusted using a Tukey-Kramer adjustment.
Northern analysis
Total RNA was isolated from freshly harvested testes or purified
cells by guanidinium-isothiocyanate homogenization and centrifugation
through a cesium chloride cushion (39). The RNAs were electrophoresed
on 1% 3-(N-morpholino)propanesulfonic acid-formaldehyde
agarose gels with 10 µg total RNA loaded per lane. To verify lack of
Sertoli cell contamination in the Leydig cell fractions, less Sertoli
cell RNA (1–2 µg) was loaded on some Northerns to allow overexposure
of membranes hybridized with the androgen-binding protein probe.
Northern transfer and hybridization were performed as previously
described (5) using a full-length rat MIS type II receptor riboprobe
(4). The membranes were also hybridized with probes for
androgen-binding protein (30) and LH (33) and FSH receptors (32). To
control for sample loading and transfer, the gels and membranes were
stained with ethidium bromide and rehybridized with GAPDH (40).
Densitometric analysis of the autoradiograms was performed using NIH
Image software. To better characterize the changes in MIS expression
during pubertal maturation of the testis, Northern analysis was
performed with total RNA extracted from 14- to 60-day rat testes and
hybridized with an MIS complementary DNA probe (31).
Flow cytometric analysis of MIS binding
Flow cytometry studies were conducted with biotinylated
recombinant human MIS. Immunoaffinity purified MIS was conjugated with
biotin using a 40-fold molar excess of NHS-LC biotin (Pierce Chemical Co., Rockford, IL) in PBS on ice for 30 min at
neutral pH. Unreacted biotin was quenched with 100 mM Tris
buffer. The biotinylated MIS was separated from unconjugated MIS on a
5-ml avidin affinity column (Ultralink, Pierce Chemical Co.) by washing with 10 ml of 0.02 M Tris buffer pH
7.4, then eluted in Tris buffer containing 20 mM biotin.
The biotinylated MIS was desalted and reconcentrated, then quantitated
by Bradford assay (Bio-Rad Laboratories, Inc.) and ELISA
(41) and tested for retention of bioactivity in the rat urogenital
ridge assay (37). The degree of biotinylation of MIS was measured as a
function of dye binding (42). The biotinylated MIS was also examined by
Western analysis using streptavidin conjugated to horseradish
peroxidase and enhanced chemiluminescence (ECL, Renaissance, NEN Life Sciences Products, Inc., Boston, MA) to ensure that
it remained intact.
Flow studies were conducted with either freshly isolated Leydig or Sertoli cells, or cells that had been maintained overnight at 4 C in Leydig cell culture media. All incubations and washes were performed at 4 C or on ice to avoid receptor internalization. The cells were resuspended at 106 cells/ml in either binding buffer (0.5% BSA, 150 mM NaCl, 10 mM MgCl2, 10 mM KCl, 20 mM HEPES, pH 7.4) or Leydig cell media, then incubated with the appropriate concentration of biotinylated MIS, or buffer alone for 1 h. The cells were washed in binding buffer or Leydig cell media, then resuspended in PBS and incubated with 7 µg avidin phycoerythrin conjugate (Molecular Probes, Inc., Eugene, OR) for 30 min in the dark. Unbound avidin phycoerythrin was washed off with PBS before the cells were resuspended in 250 µl PBS for flow analysis, using a Becton Dickinson and Co. FACScan flow cytometer. For competition experiments, either nonbiotinylated MIS (10-fold excess) or TGF-ß1 (Promega Corp.; up to 40 nM) were coincubated with the biotinylated MIS at select concentrations. Competition was not performed at all MIS doses to conserve the limited supplies of recombinant human MIS.
Before analysis, the FACScan flow cytometer was calibrated for fluorescence and light scatter sensitivity with Calibright beads and AutoComp software (Becton Dickinson and Co.) to ensure day-to-day analytic consistency. Calibright beads were run with BD Autocomp software using the photomultiplier, compensation, and sensitivity software options. Forward and side angle light scatter settings were optimized and standardized for Leydig and Sertoli cells. Stained cells were applied to the flow cytometer, with LYSYS II acquisition and analysis software. Forward vs. side angle scatter gating and propidium iodide exclusion was used to identify viable cells from dead cells and debris, with 10,000 events collected in list mode over 1024 channels/parameter for each sample. Once the machine was calibrated for the day, no further adjustments were made during subsequent analyses performed on that day.
Mean and median fluorescence per cell were plotted against the MIS concentration to generate saturation binding curves, which were fitted by a polynomial regression algorithm. The dissociation constant of the MIS-receptor complex was calculated as the concentration of biotinylated MIS needed for 50% saturation of binding sites. Nonspecific binding was determined by the amount of binding remaining in the presence of 750 nM nonbiotinylated MIS. Nonspecific binding was subtracted from total binding to estimate specific binding.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and
increased in more mature ILCs (Fig. 1B), as previously observed (19).
The Sertoli cell enriched fractions contained 0.5–1% germ cells that
failed to attach after overnight culture in serum-free media and were
removed with a media change. The Sertoli cells did not stain for
3ß-hydroxysteroid dehydrogenase. To further assess the purity of each
cellular fraction, Northern analysis was performed with cell-type
specific probes using replicate RNAs isolated from different cell
preparations (n = 3–8). These confirmed that LH receptor mRNA was
present only in Leydig cells and total testis, whereas Sertoli
cell-specific MIS, FSH receptor, and androgen binding protein mRNAs
were detected primarily in Sertoli cells and total testis, but not in
Leydig cells (Table 1).
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). At both 21 and 35 days of age, the
Leydig cells predominantly expressed a shorter MIS type II receptor
transcript, in contrast to the Sertoli cells, which also expressed the
longer transcript present in whole testis. The steady-state mRNA levels
of the MIS type II receptor in Sertoli cells were more abundant on day
21 than day 35, when the signals were normalized for GAPDH by
densitometry (Table 2). In contrast, the
abundance of the receptor transcripts in Leydig cells was comparable at
day 21 (PLCs) and 35 (ILCs).
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and Table 2).
Decreasing the amount of Sertoli cell RNA loaded enabled a longer
exposure to be obtained without obscuring the Leydig cell lanes and
confirmed the absence of ABP signal in PLCs and ILCs.
Rehybridization with GAPDH, a ubiquitous gene, demonstrated that the
total testis and Leydig cell lanes contained comparable amounts of RNA
(Fig. 2). Quantitation of the autoradiograms by densitometry indicated
that the abundance of GAPDH varied by less than 2-fold in all lanes
(998 to 1865 arbitrary units) except for the two Sertoli cell lanes.
The film intensity signal of the 21-day-old Sertoli cell lane was about
5-fold less (237 arbitrary units) and that of the 35-day Sertoli cell
lane was about 30-fold less (32 U) than the other lanes.
Steady-state MIS mRNA levels in whole testis declined postnatally but were transiently induced in the peripubertal rat testis from days 14 to 20 (data not shown), coincident with induction of MIS type II receptor expression in the postnatal testis. By day 24, MIS mRNA levels had again declined and continued to decrease in the maturing testis. This was consistent with previously published data showing a transient increase in MIS immunoreactivity in the 20 day rat testis (23).
MIS binding to Leydig cells
PLCs bound biotinylated MIS specifically as measured by an
increase in mean and median fluorescence per cell (of 10,000 events
recorded per condition) in the presence of increasing concentrations of
MIS (Fig. 3A). A series of ten individual
flow studies conducted with different PLC preparations consistently
demonstrated shifts in fluorescence with biotinylated MIS. At 30
nM, the increase in mean fluorescence per cell ranged from
15–100% above baseline. Ten-fold excess nonbiotinylated MIS
specifically competed for binding and shifted the increased
fluorescence with 30 nM biotinylated MIS back to baseline.
Similar competition was seen at 75 nM MIS with excess
nonbiotinylated MIS (750 nM).
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). At 35 days of age, neither ILCs nor Sertoli cells
exhibited a shift in mean fluorescence in the presence of increasing
doses of biotinylated MIS (data not shown).
The binding visualized by flow cytometry was concentration dependent
from 10 to 150 nM and saturable (Fig. 4). The concentration of MIS required to
reach half saturation was approximately 15 nM, a measure of
the dissociation constant of the MIS-receptor complex. Nonspecific
binding determined by competition with nonbiotinylated MIS at selected
doses accounted for about 30–40% of the total binding. To ascertain
that MIS was not binding to the TGF-ß receptor, the cells were
coincubated with TGF-ß at 1,000 to 10,000-fold molar excess of its
ED50 (0.4–40 nM). Excess TGF-ß did not
compete for binding, indicating that MIS was binding to its own
receptor rather than to the TGF-ß receptor (Fig. 4). The saturation
binding curve and dissociation constant of PLCs were similar to that
seen in flow studies with several transformed ovarian cancer cell lines
that express the MIS type II receptor (Masiakos, P. T., D. T.
MacLaughlin, J. T. Teixeira, S. Maheswaran, P. C. Shah,
D. J. Kehas, M. K. Kenneally, D. M. Dombkowski, T.
U. Ha, F. I. Preffer, and P. K. Donahoe, submitted).
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. With increasing concentrations of MIS,
PLCs exhibit a progressive increase in the mean fluorescence per cell,
whereas neither primary Sertoli cells nor COS cells show any shift.
Again, competition with excess nonbiotinylated MIS decreased the mean
fluorescence of PLCs back to baseline, but had no effect in the Sertoli
cells. Because no fluorescent shift was demonstrated with COS cells,
competition was not performed.
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). In the absence of LH, only the
highest MIS concentration of 43 nM significantly decreased
thymidine incorporation from a mean cpm/million cells of 818 to 433
(P < 0.05, n = 4). The decrease in thymidine
incorporation to 73% of control with 14 nM MIS was not
statistically significant. Addition of low dose LH (1 ng/ml) to the
culture media enhanced the basal incorporation of thymidine in the
buffer control wells from 1.5- to 5-fold in individual experiments
(mean basal cpm/million cells increased from 818 to 2146) and
potentiated the effect of lower concentrations of MIS. In the presence
of LH, 3.5 to 43 nM MIS caused inhibition of thymidine
incorporation that varied from 50–64% of control (P
< 0.001, n = 7). Within this concentration range, no dose effects
were detectable by Tukey analysis.
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). The apparent increased thymidine
incorporation with added MIS by ILCs cultured in the absence of LH was
not statistically significant. The addition of low dose LH did not
confer responsiveness to MIS (n = 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lack of binding of MIS to Sertoli cells despite abundant MIS type II receptor mRNA is consistent with transgenic and knock-out models of MIS that have no readily discernable Sertoli cell phenotype (12, 13). Additional studies to further characterize developmental changes in the expression and signaling of MIS receptors in Sertoli cells are necessary to determine whether MIS has an unrecognized physiologic function in the Sertoli cell.
These findings support our hypothesis that MIS has a direct role in the control of testicular development. Prepubertally, MIS is present, but its receptor expression is low or negligible in the testis (4), enabling the mesenchymal Leydig cell precursors to proliferate. By day 21, the mesenchymal stem cells attain the progenitor stage of differentiation and start to express steroidogenic enzymes and to decrease their rate of proliferation (19, 21). At this stage of development, MIS transcription appears to be transiently induced in Sertoli cells, and the type II receptor is readily detected in the PLCs. Thus, perhaps MIS serves to limit the proliferation of PLCs and to promote their differentiation and maturation into more steroidogenically active cells. By day 35, PLCs have differentiated into ILCs that only undergo one more cycle of cell division (19, 21). The differentiated state and low proliferative index of ILCs may obviate the need for the antiproliferative effects of MIS at this stage of Leydig cell maturation. In the absence of MIS, as exemplified in mice with null deletions of the MIS ligand and/or its receptor, the Leydig cells become hyperplastic and develop foci of neoplasia (11, 12).
Leydig cell proliferation and steroidogenesis are under the regulation of diverse hormones and growth factors (43, 44). Insulin-like growth factor I (IGF-I), thyroid hormone, LH, and Sertoli cell paracrine factors are all proliferative stimuli for PLCs and ILCs (22, 44, 45, 46). The same panel of hormones and growth factors can promote differentiation and enhance steroidogenic capacity of Leydig cells, with LH playing a predominant role in stimulating testosterone biosynthesis (20, 21, 47, 48). MIS may be a previously unrecognized player in the regulation of Leydig cell maturation and differentiaton by limiting the proliferation of PLCs. Moreover, this antiproliferative effect of MIS appears to be occurring independently of LH, as the expression of LH and its receptor are unchanged in mice with null deletions of the MIS gene (24). Although this study has demonstrated that MIS decreases DNA synthesis in the Leydig cells, the underlying mechanism remains unclear. Future studies will focus on clarifying the molecular basis of this inhibitory effect to determine whether MIS is causing cell cycle arrest or increasing apoptotic cell death of the PLCs.
MIS decreases net testosterone production (24, 25) by down-regulating steady-state mRNA levels of steroidogenic enzymes in testes from 2-month-old mice (24) and in R2C and MA-10 cell lines of Leydig cell origin (Teixeira, J., A. H. Payne, and P. K. Donahoe, unpublished data). MIS also inhibits activity of a P450c17/luciferase transgene in MA-10 cells, suggesting that MIS may be affecting transcription of this gene to regulate testosterone synthesis (Teixeira, J., A. H. Payne, and P. K. Donahoe, unpublished data). Determinants of testicular testosterone production, however, include both the steroidogenic capacity of individual Leydig cells and the total number of Leydig cells per testis. Our data indicate that MIS limits Leydig cell number in addition to its reported effects on steroidogenesis. This action of MIS would help prevent unregulated expansion of the Leydig cell population and control excessive androgen production during early puberty to achieve a normal progression of puberty.
Negative growth regulators are increasingly being recognized as having critical roles in balancing cell proliferation and cell death. Therefore, a better understanding of the mechanism by which MIS prevents hyperplastic growth and neoplastic transformation of Leydig cells will offer additional insights into the regulation of testicular development and tumorigenesis. Mice with targeted deletions of the MIS ligand or receptor develop neoplasms that resemble human Leydig cell tumors (11, 12). These tumors secrete either testosterone, causing isosexual precocity (49), or other atypical sex steroids, including estrogens, causing prepubertal gynecomastia or discordant (feminizing) secondary sexual characteristics at puberty (50). The secretion of excessive sex steroids not only produces sexual precocity but also causes early skeletal fusion and compromises linear growth. The treatment of these tumors by orchidectomy results in further morbidity, impairing secondary sexual maturation, future fertility, and sexual functioning. The elucidation of a role for MIS in the prevention of testicular tumorigenesis may potentially yield alternative therapeutic modalities for the management of these patients.
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Acknowledgments |
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Footnotes |
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2 Currently a Ph.D. student in the Department of Pathology, Boston
University School of Medicine, Boston, Massachusetts.
Received October 23, 1998.
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), the mean
fluorescence per cell did not change significantly.
