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Effect of deprival of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and IGF-1
Molecular and Cellular Endocrinology 162 (2000) 113 – 120 www.elsevier.com/locate/mce
Effect of deprival of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and IGF-1 V. Sriraman a, Veena S. Rao b, M.R. Sairam c, A. Jagannadha Rao a,b,* b
a Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Department of Molecular Reproduction, De6elopment and Genetics, Indian Institute of Science, Bangalore 560 012, India c Clinical Research Institute of Montreal, Montreal, Que´bec, Canada PA H2W1 R7
Received 20 October 1999; accepted 21 January 2000
Abstract The levels of proliferating cell nuclear antigen (PCNA) and cyclin D3 which are known markers of cellular proliferation were monitored by immunoblotting in progenitor Leydig cells (PLC), immature Leydig cells (ILC) and adult Leydig cells (ALC) isolated from 21, 35 and 90 day old rats, respectively which represent the Leydig cells at different stages of development. The levels of PCNA and cyclin D3 were highest in PLC, intermediate in ILC and lowest in ALC. Following administration of an antiserum to LH to deprive endogenous LH in 21 day old rats, a significant decrease in the levels of PCNA and Cyclin D3 were observed suggesting the involvement of Lutenizing hormone (LH) in PLC proliferation. In support of this observation, Bromodeoxyuridine (BrdU) incorporation was highest in PLC when compared with ILC and ALC, and administration of LH antiserum to 21 day old rats led to a total absence of BrdU incorporation by the isolated PLC. Also, there was a decrease in the level of IGF-1 and IGF-1 receptor mRNA levels by 55 and 35%, respectively as assessed by semi-quantitative RT-PCR. In addition, the PLC isolated from rats deprived of endogenous LH incorporated much less BrdU following addition of IGF-1. These results, which are obtained using an in vivo model system establish that LH has a very important role in Leydig cell proliferation in immature rats. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Leydig cells; Lutenizing hormone; Proliferating cell nuclear antigen; Cyclin D3; IGF-1; IGF-1 receptor
1. Introduction The post-natal development of Leydig cells in rat involves its proliferation, morphological differentiation and acquisition of the capacity to produce testosterone by passing through three different stages of development. Formation of adult Leydig cells involves transformation of mesenchymal like cells to Leydig cell progenitors by day 21 which has low levels of LH receptors and has less steroidogenic capacity (Benton et al., 1995). Further, they differentiate into immature Leydig cells by day 35 and during this process they acquire steroidogenic organelle structure and enzymatic capacities, to synthesize and metabolize testosterone. The level of LH receptor in these cells are more than PLC and as ILC differentiate to adult Leydig cells they * Corresponding author. Fax: +91-80-3345999. E-mail address: [email protected] (A.J. Rao)
predominantly synthesize testosterone and possess highest levels of LH receptor (Benton et al., 1995). In all cells proliferation is controlled by molecules positively or negatively affecting the kinase cascades that regulate transitions through checkpoints of the cell cycle. Entry into cell cycle is positively regulated by D-cyclins — D1, D2 and D3 (Inaba et al., 1992; Xiong et al., 1992a). The D-cyclins binds to cyclin-dependent kinases and this active complex phosphorylates cellular substrates resulting in DNA synthesis and ultimately enables the cell to enter into S-phase from G1-phase. Of the various proteins involved in DNA synthesis PCNA, a 36 kDa protein is an essential component that binds to DNA polymerase d and D-cyclins to initiate cell cycle progression (Laskey et al., 1989; Liu et al., 1989; Xiong et al., 1992b). Hence, analysis of DNA synthesis by BrdU incorporation and assessing the levels of D-cyclins (Robker and Richards, 1998) and PCNA (Bravo and Macdonald-Bravo, 1987; Hall et al.,
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1990; Ogle et al., 1998) at different stages of post-natal development of Leydig cell would reflect its proliferative capacity. LH has been found to be indispensable for functional differentiation of adult Leydig cells, but the precise role of LH in Leydig cell proliferation is not clear. Since serum LH levels do not rise with the onset of puberty (Dohler and Wuttke, 1975) and mesenchymal like stem cells proliferate in hypophysectomized animals after EDS treatment, it is suggested that LH is not required for proliferation of Leydig stem cells (Benton et al., 1995). In addition, it was also reported that LH does not induce marked DNA synthesis in immature Leydig cells in vitro, and hence LH is considered to be less involved in stimulating Leydig cell proliferation (Khan et al., 1992a,b; Ge et al., 1996). However, Christensen and Peacock (1980) demonstrated that following administration of hCG to adult rats for 5 weeks there was an increase in Leydig cell number though no increase was seen during the first 3 weeks. In contrast, Kuopio et al. (1989) reported that only foetal neonatal Leydig cells exhibit rapid proliferative response to LH. By monitoring a direct biochemical parameter such as increase in the 3H thymidine incorporation by the Leydig cells from adult rats treated with hCG, Abney and Carswell (1986) showed that LH/hCG, stimulated proliferation of Leydig cells 48 h after hCG administration. However, this conclusion has been questioned by Moore et al. (1992) on the ground that Leydig cells that were used for proliferation studies by Abney and Carswell (1986) were only partially pure and thus do not reflect proliferation of only Leydig cells. Thus, it appears although one can demonstrate the proliferation of Leydig cells following LH administration, the precise regulatory events that occur under the influence of LH in Leydig cell proliferation and the absolute need of LH for this process have not been established. In addition, IGF-1 is an important growth factor which promotes Leydig cell differentiation and proliferation and its testicular level increases with puberty (Closset et al., 1989). Leydig cells of mice with a targeted deletion of IGF-1 gene remain functionally immature, showing the requirement of IGF-1 in Leydig cell development (Benton et al., 1995). Hence, analysis of regulation of the above said cell cycle regulatory molecules under conditions of LH deprival will provide a better understanding of the role of LH in Leydig cell proliferation. In addition analysis of IGF-1 and IGF-1 receptor mRNA levels, and assessment of IGF-1 stimulated BrdU incorporation following LH deprival would provide an insight into the role of LH in regulation of IGF-1 mediated proliferation of Leydig cells. Towards this, passive neutralization studies were carried out after ascertaining the period at which Leydig cells exhibit a maximum proliferative capacity. In this approach we have employed a highly specific antiserum to ovine LH capable of neutralizing endogenous LH in rats for a selected duration.
2. Materials and methods
2.1. Chemicals Collagenase and DNAse for cell isolation were procured from Worthington, NJ. Bovine Serum Albumin (BSA), Soya Bean Trypsin Inhibitor, DMEM-Ham F-12, IGF-1, Percoll, anti-mouse IgG peroxidase conjugate and TRIzol were obtained from Sigma, St. Louis, MO. Antibodies to proliferating cell nuclear antigen and D-cyclins (cyclin D1, D2, D3) were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. MMLV-Reverse transcriptase, dNTP’s, cell proliferation ELISA system and ECL system were procured from Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK. Taq Polymerase and primers for PCR were obtained from Bangalore Genei, Bangalore, India. Rat LH and hCG was a kind gift from Dr A.F. Parlow, National Hormone & Pituitary Program, NIDDK, USA. Highly purified ovine LH was prepared at Clinical Research Institute of Montreal, Montreal.
2.2. Animals Wistar rats of 19, 35 and 90 days were obtained from the National Institute of Nutrition, Hyderabad, India and maintained under standard conditions (12 h of light and 12 h of dark schedule, with water and pelleted food ad libitum). Animal procedures employed in the study were cleared by institutional ethical committee.
2.2.1. LH antiserum treatment The antiserum to highly purified ovine LH was raised in adult bonnet monkeys which at a dilution of 1:20 000 bound 75% of iodinated rat LH. The ability of this antiserum to neutralize endogenous LH in rats was established by demonstrating that a single injection of 275 ml of the antiserum to adult male rats (90 days) reduced serum testosterone concentration to 12% of value seen in the control animals that received an equal volume of pre-immune serum (Table 1). Serum testosterone levels were determined by specific radioimmunoassay standardized in the laboratory (Rao and Kotagi, 1989). Immature male rats (19 days old) received 125 ml LH a/s for two days (days 19 and 20) by subcutaneous injection and were sacrificed on day 21 for PLC isolation. Animals which received equal volume of pre-immune monkey serum served as control. 2.3. Isolation of PLC, ILC and ALC Cells were isolated from different age groups of rats, according to the procedure of Klinefelter et al. (1987) and Hardy et al. (1990). Adult Leydig cells were purified by elutriation followed by Percoll density gradient fractionation. In view of the fact there is minimal
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spermatogenic activity in 21 and 35 day old rats only density gradient fractionation of interstitial cells was used for isolation of PLC and ILC. The density gradient was formed by centrifugation at 20 000× g at 4°C for 1 h, PLC were collected between densities of 1.064– 1.070. Similarly, for ILC and ALC fractions between 1.070 and 1.088 were collected. Cells were washed with DMEM-Ham F12, pelleted at 250 × g, and resuspended in DMEM-Ham F12 and used for further studies.
2.4. Cell culture Purified cells (PLC, ILC, ALC) were plated in 96well culture plates (20 000 cells/well in 200 ml DMEMF12 containing 0.1% BSA and 12 mg/ml gentamycin, referred to as culture medium) at 34°C. Cells were allowed to attach for the first 15 h, followed by replacement of the culture medium (200 ml) with or without 100 ng of hCG or 15 ng of IGF-1. Cells were incubated in culture medium containing 10 mM BrdU for 12 h after addition of hCG and 12 h later; cells were processed for determining the incorporation of BrdU by ELISA according to the manufacturer’s instructions.
2.5. Western blotting of PCNA and D-cyclins Leydig cells were homogenized in 50 mM PBS and the solubilised proteins were estimated by Lowry’s method using BSA as standard (Lowry et al., 1951). Fifty mg of the solubilised proteins were subjected to SDS-PAGE (10%) and Western blotting was carried out, according to the procedure of Towbin et al. (1979). Following incubation of the membranes with rat monoclonal antibody to D-cyclins (D1, D2 and D3) or PCNA in 5% aqueous solution of non-fat dry milk powder at a dilution of 1:500, membranes were washed and incubated with rabbit anti-mouse IgG conjugated to horse radish peroxidase. After washing, the membranes were developed to visualize the immunoreactive band using ECL detection system. Intensity of signal was quantitatively measured by densitometry of the films. Table 1 Reduction in serum testosterone levels by LH antiseruma Serum testosterone ng/ml (mean 9SE) Pre-immune 2.72 90.47 serum-treated LH 0.337 90.04 antiserum-treat ed
2.6. Semi-quantitati6e RT-PCR for IGF-1 and IGF-1 receptor Total RNA was isolated from progenitor Leydig cells using TRIzol reagent and intact RNA with A260/280 ratio 1.6 above was used for RT-PCR. One mg of RNA was reverse transcribed using 50 units MMLV-reverse transcriptase with random hexamers in a 20 ml reaction volume for 1 h at 37°C. An aliquot of 2 ml from the RT mixture containing the cDNAs was used for PCR in a 50 ml of reaction volume. The oligonucleotide primers for amplifying IGF-1 were 5%-GCC ACA GCC GGA CCA GAG ACC CTT-3% and 5%-CTA CAT TCT GTA GGT CTT GTT TCC-3% and the oligonucleotide sequence to amplify IGF-1 receptor were 5%-CTG TGT TCT TCT ATG TCC CA-3% and 5%CGA GCT CCC GGT TCA TGG TG-3% (Murphy et al., 1987; Kurachi et al., 1992). Cyclophilin was employed as internal control and primers used to amplify were 5%-GTG GCA AGT CCA TCT ACG-3% and 5%-CAG TGA GAG CAG AGA TTA CAG-3% (Majdic et al., 1996). The expected size of the products were 327 bp, 261 and 380 bp for IGF-1, IGF-1 receptor and cyclophilin, respectively. The PCR was performed within the linear range of amplification to assess their expression levels with cyclophilin as an internal control. The number of cycles performed for IGF-1 and IGF-1 receptor were 30, and 28 for cyclophilin. After an initial denaturation at 94°C for 3 min each cycle included 1 min at 95°C, 1 min at 56°C and 1 min at 72°C except that for IGF-1 receptor the annealing temperature was 52°C. The PCR amplification was carried out for respective number of cycles as stated above and 12 ml of each RT-PCR product was electrophoresed in 2% agarose gel with Ethidium Bromide in 0.045 M Tris–borate/0.001 M EDTA buffer. The difference in intensities of the product following electrophoretic analysis was analysed using EDAS 120 Kodak Gel documentation system.
2.7. Statistical analysis The data were analysed by Kruskal–Wallis ANOVA and a P value less than 0.05 was considered to be statistically significant. The data presented in figures and graphs were representative of at least three independent experiments.
Percentage inhibition (%) – 87.7
LH antiserum (275 ml) was administered to adult male rats (n=6/group) by intraperitoneal route and 24 h later serum testosterone was determined by specific RIA. a
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3. Results
3.1. Purity and functional characteristics of PLC, ILC and ALC The purity of the cells isolated form different age groups of animals was assessed by 3b-hydroxysteroid dehydrogenase histochemical staining PLC and ILC
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Fig. 1. Western blot analysis of cyclin D3 protein in Leydig cells. The homogenates of PLC (lane 1), ILC (lane 2), ALC (lane 3) containing 50 mg of protein were electrophoresed and transferred to nitrocellulose membrane for detection of 30–34 kDa cyclin D3 protein with a mouse monoclonal antibody at a dilution of 1:500 by ECL system (Amersham).
Fig. 2. Western blot analysis for PCNA protein in Leydig cells. The homogenates of PLC (lane 1), ILC (lane 2), ALC (lane 3 represents cells isolated by Percoll gradient fractionation and lane 4 represents cells isolated by elutriation followed by Percoll gradient fractionation) containing 50 mg of protein were electrophoresed and transferred to nitrocellulose membrane for detection of the 36 kDa PCNA protein with a mouse monoclonal antibody at a dilution of 1:500 by ECL system (Amersham).
hCG (100 ng) as monitored by in vitro testosterone production was also assessed. It is to be noted that although the major end product of steroidogenic response by PLC is 5a-androstane-3a,17b-diol and not testosterone as in the case of ALC, still an increase in testosterone can be observed following in vitro incubation of PLC with hCG/LH (ng of testosterone produced/106 cells for 3 h) mean9 SD, n=3 with PLC, control 0.19 0.02, hCG 0.239 0.03 with ALC; control 3.190.1, hCG 25.291.2).
3.2. D-cyclins in Leydig cells The presence of D-cyclins (D1, D2 and D3) were assessed in PLC, ILC and ALC since their association with cyclin-dependent kinase forms an active complex leading to phophorylation of various intracellular substrates resulting in activation of genes involved in DNA synthesis and ultimately enables cells to begin the transition from G1- to S-phase. Western blotting of cyclin D1, D2 and D3 in the samples of purified Leydig cells revealed an absence of cyclin D1 and D2 (data not shown) and presence of cyclin D3, consistent with the known molecular weight 32 kDa. The level of cyclin D3 decreased with increasing age and when the levels of cyclin D3 were expressed relative to PLC, it was observed that while it was 809 3% in the ILC, and was only 3.469 2% in the case of ALC (Fig. 1).
3.3. PCNA in Leydig cells PCNA, a 36 kDa protein is an essential replication factor and hence its immunoreactivity is a useful marker for cell proliferation and its level correlates with other parameters of proliferation. An immunoblot of the Leydig cells proteins, for PCNA revealed an age-dependent reduction in its level as the Leydig cells lose their proliferative capacity and differentiate. When the PCNA levels were expressed relative to PLC it was only 1592% in ILC and 7.49 1.5% in ALC (Fig. 2).
3.4. Effect of hCG on in 6itro Leydig cell proliferati6e capacity
Fig. 3. Effect of addition of hCG on BrdU incorporation in culture in Leydig cells. PLC, ILC and ALC were isolated and cultured as described in Section 2. Bars corresponding to 1, 3, 5 and 2, 4, 6 indicate the BrdU incorporation values without and with hCG. Values represent mean 9SE from three separate experiments cultured in triplicates. Statistical significance tested between groups 2 and 4 and groups 2 and 6 were **P= 0.0082.
were found to be 92% pure and ALC were 90% pure. The response of these cells to a fixed concentration of
The proliferative capacity during the course of postnatal development of Leydig cells with hCG was assessed by DNA synthesis during culture by monitoring BrdU incorporation. Following addition of hCG a significant increase in BrdU incorporation in PLC (0.8 O.D./20 000 cells) was noted. However, it was significantly less in ILC (0.075 O.D./20 000 cells) and completely absent in ALC (Fig. 3). These results indicate that LH stimulates DNA synthesis in PLC and there is a progressive loss in the proliferative capacity of cells isolated from animals of increasing age.
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3.5. Effect of depri6al of endogenous LH on bromodeoxyuridine incorporation, PCNA and cyclin D3
Fig. 4. Effect of in vivo LH deprival on BrdU incorporation by PLCs in culture. PLCs were isolated from Pre-immune serum (PIS) and LH-antiserum (LH a/s) treated groups and cultured as described in Section 2. Values represent mean 9 SE. Data shown from at least three separate experiments (***P=0.0002).
Fig. 5. Western blot analysis of Leydig cell cyclin D3 after LH antiserum treatment. The homogenates of PLC from pre-immune serum-treated (lane 1) and LH antiserum-treated (lane 2) groups containing 50 mg of protein were electrophoresed and transferred to nitrocellulose membrane for detection of 30–34 kDa cyclin D3 protein, with a mouse monoclonal antibody at a dilution of 1:500 by ECL system (Amersham).
In order to ascertain the role of LH in modulation of these regulatory proteins, a passive neutralization study was carried out in immature rats (19 days old), since Leydig cells isolated from this period showed a maximum proliferative capacity. Deprival of endogenous LH for 2 days (days 19 and 20) using a specific antiserum to LH led to total absence of BrdU incorporation when isolated Leydig cells were cultured in the presence of hCG, unlike in the control group in which an 8-fold increase was seen (Fig. 4). When Leydig cells proteins were analyzed by Western blot for cyclin D3 and PCNA the level of cyclin D3 in the LH antiserumtreated group was much smaller when compared with that in the control (Fig. 5). Similarly, the level of PCNA was found to be decreased by 60% in the LH antiserum-treated group (Fig. 6). It has been well-documented that in vitro addition of IGF-1 stimulates DNA synthesis in progenitor Leydig cells (Ge and Hardy, 1997), and our results reveal that when IGF-1 stimulated DNA synthesis was assessed by BrdU incorporation in cells isolated from LH a/s treated rats, there was a significant decrease in incorporation (data not shown). These results indicate that specific deprivation of LH leads to decreased DNA synthesis as assessed by BrdU incorporation and decrease in the levels of cyclin D3 and PCNA proteins which play a key role in G1 to S transition and DNA replication.
3.6. PCR based quantitation of IGF-1 and IGF-1 receptor mRNA le6els Several studies have established an important role for IGF-1 in control of Leydig cell proliferation (Benton et al., 1995). Since our studies revealed an important role for LH and there was loss of IGF-1 and IGF-1 receptor in Leydig cells following deprival of endogenous LH in 21 day old rats. A semi-quantitative RT-PCR analysis revealed a 52.5 9 2.5% reduction in mRNA levels of IGF-1 and 389 2% decrease in IGF-1 receptor (Fig. 7) when expressed relative to cyclophilin. These results suggests that LH is necessary to maintain the mRNA levels for IGF-1 and IGF-1 receptor.
4. Discussion
Fig. 6. Western blot analysis of PCNA in Leydig cells after LH antiserum treatment. The homogenates of PLC from pre-immune serum-treated (lane 1) and LH antiserum-treated (lane 2) groups containing 50 mg of protein were electrophoresed and transferred to nitrocellulose membrane for detection of the 36 kDa protein, with a mouse monoclonal antibody at a dilution of 1:500 by ECL system (Amersham).
Our results demonstrate a decrease in cyclin D3 and PCNA along with a loss in proliferative capacity as a function of age in Leydig cells and also following deprival of endogenous LH in immature rats. In this connection it is pertinent to note that in a recent study it has also been reported that expression of cyclin D3 is strong in endocrine cells secreting steroid hormones and
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Fig. 7. Semi-quantitative RT-PCR for IGF-1 and IGF-1 receptor in progenitor Leydig cells following deprival endogenous LH. RNA isolated purified PLCs were reverse transcribed, cDNA obtained was subjected to a semi-quantitative PCR in the linear range of amplification for (a) IGF-1; (b) IGF-1 receptor and (c) cyclophilin as an internal control. Lanes 1 and 2 correspond to PCR amplified products from pre-immune serum-treated and LH antiserum-treated groups, respectively.
in normal human testes, the cell type-restricted expression patterns were dominated by high levels of cyclin D3 in Leydig cells (Doglioni et al., 1998; Bartkova et al., 1999). Considering this it is appropriate that we have monitored the levels of cyclin D3 as an indicator for proliferative activity of progenitor Leydig cells. The observation that the incorporation of BrdU which is an indicator of DNA synthesis is maximum only in PLC isolated on day 21 when compared with cells isolated on days 35 and 90 suggests that proliferative capacity is maximum in PLC and this age group is the most appropriate to study the role of LH in Leydig cell proliferation. In addition, our results suggest that a sharp decline of cyclin D3 and PCNA in immature and adult Leydig cells could contribute to their loss of proliferative capacity since these proteins mediate the G1 to S transition and DNA synthesis. It is well-known that D-cyclins bind to cyclin-dependent kinase (cdk) 4 or cdk 6 which allows phosphorylation by cdk-activating kinase thereby forming an active complex which phosphorylates retinoblastoma protein RB and other related proteins leading to activation of E2F family of transcription factors. This leads to the activation of numerous genes involved in DNA synthesis and ultimately enables cells to begin the transition from G1- to S-phase (Robker and Richards, 1998). PCNA is a essential replication factor synthesized at the initial stages of G1-phase and has a longer half life accumulat-
ing in the nucleus until mitosis. PCNA binds to DNA polymerase-d and D-cyclins to initiate cell cycle progression (Ogle et al., 1998). Thus immunoreactivity of D-cyclins and PCNA are useful markers of cell proliferation and this correlates with other parameters employed to assess cellular proliferation. The results obtained in the present study reveal that the levels of PCNA and cyclin D3 as assessed by Western blot, are maximum in PLC’s isolated from 21 day old rats and this correlates with their ability to synthesize DNA in vitro following addition of hCG when compared with Leydig cells isolated from day 35 (ILC) and day 90 (ALC). Several factors are known to modulate Leydig cell proliferation but the primary stimulus for the pubertal increase in Leydig cell number has not been established. Of the various factors involved LH has been found to be indispensable for functional differentiation of Leydig cell proliferation is not clear. In contrast to the earlier studies which have employed either impure preparation of Leydig cells or morphometric and histological parameters to assess the role of LH to stimulated Leydig cells proliferation, we have employed purified Leydig cells and also more than one parameter namely, BrdU incorporation, level of PCNA, and cyclin D3 to assess the role of LH in stimulation of Leydig cell proliferation. Before using the Leydig cells isolated from immature rats (21 days) to evaluate the role of LH in Leydig cell proliferation, we have ascertained that this age group is the most appropriate one for proliferation study. In addition, we have employed the procedure standardized and validated by Klinefelter et al. (1987) and Hardy et al. (1990) to isolate Leydig cells and the isolated cells were highly pure as assessed by 3b-HSD staining as well as by the demonstration that cells isolated from different age groups respond to hCG/LH and the maximum response was seen with ALC. Our results of in vitro response studies with PLC to hCG/LH provide additional support to the conclusion that LH stimulates proliferation in these cells in spite of the fact that these cells have low level of LH receptor as reported by Shan and Hardy (1992). It is also known that the circulating levels of LH are low in 18 day old rats and thus it appears that the low level of LH receptor in PLC are adequate to stimulate the proliferative response of the cells to added hCG/LH. However, ALC which have maximum levels of LH receptor do not exhibit proliferative response to added hCG/LH although the ability of LH to induce functional differentiation of ALC is well-documented in literature. Our study also employs the approach of selective neutralization of LH for only two days without the surgical trauma of the hypophysectomy or the other effects likely to be seen following administration of EDS. In addition, the usefulness of specific antibodies to investigate the role of LH in rats and monkeys
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has been well established earlier (Moudgal et al., 1974). Neutralization of LH resulted in total absence of BrdU incorporation by isolated Leydig cells in culture following addition of hCG and a drastic decrease in cyclin D3 and PCNA protein was seen as assessed by immunoblotting. The role of LH in stimulation and maintenance of smooth endoplasmic reticulum which is involved not only in biosynthesis of steroids but also in general protein synthesis (Ewing and Zirkin, 1983) has been well-documented. Long term suppression of LH is associated with Leydig cell atrophy, reduction in steroidogenic enzymes and testosterone synthesizing capacity with drastically diminished smooth endoplasmic reticulum (Wing et al., 1985). Also acute stimulation of Leydig cells with LH involves the synthesis of at least 800 – 900 new proteins (Luo et al., 1998). Hence, deprival of LH for 2 days would have resulted in a loss of cellular integrity due to reduction in cellular proteins ultimately leading to loss in proliferative capacity. It should be noted that while a complete absence of cyclin D3 was noticed, only 60% reduction in PCNA was seen and it could be due to higher half life of PCNA protein (Ogle et al., 1998) when compared with cyclin D3. This indicates that LH is necessary for inducing the changes in the levels of cyclin D3 and PCNA, and regulation of Leydig cell proliferation. Based on the results of an in vitro study, which consisted of monitoring mRNA levels of cyclin A2, cyclin G1 and 3H thymidine incorporation following addition of LH Ge et al. (1996), reported that LH is important for maintenance of PLC proliferation. However, in our present study we have monitored in vitro DNA synthesis, levels of cyclin D3 and PCNA in purified Leydig cells from immature rats deprived of endogenous LH which will reflect the in vivo role of LH in Leydig cell proliferation. Although, results of studies with either transgenic mice overexpressing LH or LH knockout mice are likely to provide additional evidence to establish a role for LH in Leydig cell proliferation, as of now there are no reports of studies with these models. Recent reports on naturally occurring mutations in humans provide evidences for the role of LH in Leydig cell proliferation. Inactivating human LH beta mutation in humans results in absence of Leydig cells (Huhtaniemi et al., 1999) and an inactivating mutation in LH receptor leads to Leydig cell hypoplasia (Laue et al., 1996). However, it is also important to note that knockout involves a permanent loss of a specific gene product. But we have employed passive neutralization which involves selective deprival of LH for a desired period to demonstrate a stage specific function. Results of our studies also reveal that passive neutralization of LH leads to loss of IGF-1 stimulated BrdU incorporation in vitro by downregulating the mRNA levels for IGF-1 and IGF-receptor. The observation that there was a decrease in the BrdU incorpora-
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tion as well as decrease in the mRNA levels of IGF-1 and IGF-1 receptor following LH deprival suggests the possibility that LH can mediate its proliferative effects by regulating IGF-1 and its receptor in Leydig cells. In conclusion, Leydig cells lose their proliferative capacity during their course of post-natal development which is associated with decline in PCNA and cyclin D3 levels. LH plays a major role in Leydig cell proliferation by maintaining the levels of these proteins in a stage specific manner and regulating the IGF-1 network.
Acknowledgements The authors wish to thank Dr M.P. Hardy, Population Council, Center for Biomedical Research, New York for critically reviewing the manuscript and for his valuable suggestions. The authors also wish to thank Prof. A.M. Dharmarajan, University of Western Australia, Perth for reviewing the manuscript and providing the PCNA antibody. Financial assistance from Council of Scientific and Industrial Research, Indian council for Medical Research, Department of Science and Technology, Department of Biotechnology, Government of India and MRC of Canada is greatly acknowledged. Rat LH and hCG used in this study were generously provided by National Hormone and Pituitary Programme, NIDDK, USA.
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Luo, L., Chen, H., Stocco, D.M., Zirkin, B.R., 1998. Leydig cell protein synthesis and steroidogenesis in response to acute stimulation by Luteinizing hormone in Rats. Biol. Reprod. 59, 263–270. Liu, Y.-C., Marraccino, R.L., Keng, P.C., Bambara, R.A., Lord, E.M., Chou, W.-G., Zain, S.B., 1989. Requirement for proliferating cell nuclear antigen expression during stages of Chinese hamster ovary cell cycle. Biochemistry 28, 2967 – 2974. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265 – 275. Majdic, G., Sharpe, R.M., O’Shaughnessy, P.J., Saunders, P.T.K., 1996. Expression of cytochrome P450 17 alpha-hydroxylase/C17– 20 lyase in the fetal rat testis is reduced by maternal exposure to exogenous estrogens. Endocrinology 137, 1063 – 1070. Moore, A., Findlay, K., Morris, I.D., 1992. In vitro DNA synthesis in Leydig and other interstitial cells of the rat testis. J. Endocrinol. 134, 247 – 255. Moudgal, N.R., Rao, A.J., Manekjee, R., Muralidhar, K., Venkataramiah, M., Sheela Rani, C.S., 1974. Gonadotropins and their antibodies. Recent Prog. Horm. Res. 30, 47 – 77. Murphy, L.J., Bell, G.I., Duckworth, M.L., Friesen, H.G., 1987. Identification, characterization, and regulation of a rat complementary deoxyribonucleic acid which encodes insulin-like growth factor-I. Endocrinology 121, 684 – 691. Ogle, T.F., George, P., Dai, D., 1998. Progesterone and estrogen regulation of rat decidual expression of proliferating cell nuclear antigen. Biol. Reprod. 59, 444 – 450. Robker, R.L., Richards, J.D., 1998. Hormone-induced proliferation and differentiation of granulosa cells. A coordinated balance of the cell cycle regulators cyclin D2 and p27kip1. Mol. Endocrinol. 12, 924 – 940. Shan, L.X., Hardy, M.P., 1992. Developmental changes in levels of Luetenizing hormone receptor and androgen receptor in rat Leydig cells. Endocrinology 131, 1107 – 1113. Towbin, H., Stohlein, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. Wing, T.Y., Ewing, L.L., Zegeye, B., Zirkin, B.R., 1985. Restoration effects of exogenous Leuteinizing hormone on the testicular steroidogenesis and Leydig cell ultrastructure. Endocrinology 117, 1779 – 1787. Xiong, Y., Menninger, J., Beach, D., Ward, D.C., 1992a. Molecular cloning an chromosomal mapping of CCND genes encoding human D-type cyclins. Genomics 13, 575 – 584. Xiong, Y., Zhang, H., Beach, D., 1992b. D type cyclins associate with associate with multiple protein kinases and the DNA replication and repair factor PCNA. Cell 71, 505 – 574.
Effect of deprival of LH on Leydig cell proliferation: involvement of PCNA, cyclin D3 and IGF-1 V. Sriraman a, Veena S. Rao b, M.R. Sairam c, A. Jagannadha Rao a,b,* b
a Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India Department of Molecular Reproduction, De6elopment and Genetics, Indian Institute of Science, Bangalore 560 012, India c Clinical Research Institute of Montreal, Montreal, Que´bec, Canada PA H2W1 R7
Received 20 October 1999; accepted 21 January 2000
Abstract The levels of proliferating cell nuclear antigen (PCNA) and cyclin D3 which are known markers of cellular proliferation were monitored by immunoblotting in progenitor Leydig cells (PLC), immature Leydig cells (ILC) and adult Leydig cells (ALC) isolated from 21, 35 and 90 day old rats, respectively which represent the Leydig cells at different stages of development. The levels of PCNA and cyclin D3 were highest in PLC, intermediate in ILC and lowest in ALC. Following administration of an antiserum to LH to deprive endogenous LH in 21 day old rats, a significant decrease in the levels of PCNA and Cyclin D3 were observed suggesting the involvement of Lutenizing hormone (LH) in PLC proliferation. In support of this observation, Bromodeoxyuridine (BrdU) incorporation was highest in PLC when compared with ILC and ALC, and administration of LH antiserum to 21 day old rats led to a total absence of BrdU incorporation by the isolated PLC. Also, there was a decrease in the level of IGF-1 and IGF-1 receptor mRNA levels by 55 and 35%, respectively as assessed by semi-quantitative RT-PCR. In addition, the PLC isolated from rats deprived of endogenous LH incorporated much less BrdU following addition of IGF-1. These results, which are obtained using an in vivo model system establish that LH has a very important role in Leydig cell proliferation in immature rats. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Leydig cells; Lutenizing hormone; Proliferating cell nuclear antigen; Cyclin D3; IGF-1; IGF-1 receptor
1. Introduction The post-natal development of Leydig cells in rat involves its proliferation, morphological differentiation and acquisition of the capacity to produce testosterone by passing through three different stages of development. Formation of adult Leydig cells involves transformation of mesenchymal like cells to Leydig cell progenitors by day 21 which has low levels of LH receptors and has less steroidogenic capacity (Benton et al., 1995). Further, they differentiate into immature Leydig cells by day 35 and during this process they acquire steroidogenic organelle structure and enzymatic capacities, to synthesize and metabolize testosterone. The level of LH receptor in these cells are more than PLC and as ILC differentiate to adult Leydig cells they * Corresponding author. Fax: +91-80-3345999. E-mail address: [email protected] (A.J. Rao)
predominantly synthesize testosterone and possess highest levels of LH receptor (Benton et al., 1995). In all cells proliferation is controlled by molecules positively or negatively affecting the kinase cascades that regulate transitions through checkpoints of the cell cycle. Entry into cell cycle is positively regulated by D-cyclins — D1, D2 and D3 (Inaba et al., 1992; Xiong et al., 1992a). The D-cyclins binds to cyclin-dependent kinases and this active complex phosphorylates cellular substrates resulting in DNA synthesis and ultimately enables the cell to enter into S-phase from G1-phase. Of the various proteins involved in DNA synthesis PCNA, a 36 kDa protein is an essential component that binds to DNA polymerase d and D-cyclins to initiate cell cycle progression (Laskey et al., 1989; Liu et al., 1989; Xiong et al., 1992b). Hence, analysis of DNA synthesis by BrdU incorporation and assessing the levels of D-cyclins (Robker and Richards, 1998) and PCNA (Bravo and Macdonald-Bravo, 1987; Hall et al.,
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1990; Ogle et al., 1998) at different stages of post-natal development of Leydig cell would reflect its proliferative capacity. LH has been found to be indispensable for functional differentiation of adult Leydig cells, but the precise role of LH in Leydig cell proliferation is not clear. Since serum LH levels do not rise with the onset of puberty (Dohler and Wuttke, 1975) and mesenchymal like stem cells proliferate in hypophysectomized animals after EDS treatment, it is suggested that LH is not required for proliferation of Leydig stem cells (Benton et al., 1995). In addition, it was also reported that LH does not induce marked DNA synthesis in immature Leydig cells in vitro, and hence LH is considered to be less involved in stimulating Leydig cell proliferation (Khan et al., 1992a,b; Ge et al., 1996). However, Christensen and Peacock (1980) demonstrated that following administration of hCG to adult rats for 5 weeks there was an increase in Leydig cell number though no increase was seen during the first 3 weeks. In contrast, Kuopio et al. (1989) reported that only foetal neonatal Leydig cells exhibit rapid proliferative response to LH. By monitoring a direct biochemical parameter such as increase in the 3H thymidine incorporation by the Leydig cells from adult rats treated with hCG, Abney and Carswell (1986) showed that LH/hCG, stimulated proliferation of Leydig cells 48 h after hCG administration. However, this conclusion has been questioned by Moore et al. (1992) on the ground that Leydig cells that were used for proliferation studies by Abney and Carswell (1986) were only partially pure and thus do not reflect proliferation of only Leydig cells. Thus, it appears although one can demonstrate the proliferation of Leydig cells following LH administration, the precise regulatory events that occur under the influence of LH in Leydig cell proliferation and the absolute need of LH for this process have not been established. In addition, IGF-1 is an important growth factor which promotes Leydig cell differentiation and proliferation and its testicular level increases with puberty (Closset et al., 1989). Leydig cells of mice with a targeted deletion of IGF-1 gene remain functionally immature, showing the requirement of IGF-1 in Leydig cell development (Benton et al., 1995). Hence, analysis of regulation of the above said cell cycle regulatory molecules under conditions of LH deprival will provide a better understanding of the role of LH in Leydig cell proliferation. In addition analysis of IGF-1 and IGF-1 receptor mRNA levels, and assessment of IGF-1 stimulated BrdU incorporation following LH deprival would provide an insight into the role of LH in regulation of IGF-1 mediated proliferation of Leydig cells. Towards this, passive neutralization studies were carried out after ascertaining the period at which Leydig cells exhibit a maximum proliferative capacity. In this approach we have employed a highly specific antiserum to ovine LH capable of neutralizing endogenous LH in rats for a selected duration.
2. Materials and methods
2.1. Chemicals Collagenase and DNAse for cell isolation were procured from Worthington, NJ. Bovine Serum Albumin (BSA), Soya Bean Trypsin Inhibitor, DMEM-Ham F-12, IGF-1, Percoll, anti-mouse IgG peroxidase conjugate and TRIzol were obtained from Sigma, St. Louis, MO. Antibodies to proliferating cell nuclear antigen and D-cyclins (cyclin D1, D2, D3) were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. MMLV-Reverse transcriptase, dNTP’s, cell proliferation ELISA system and ECL system were procured from Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK. Taq Polymerase and primers for PCR were obtained from Bangalore Genei, Bangalore, India. Rat LH and hCG was a kind gift from Dr A.F. Parlow, National Hormone & Pituitary Program, NIDDK, USA. Highly purified ovine LH was prepared at Clinical Research Institute of Montreal, Montreal.
2.2. Animals Wistar rats of 19, 35 and 90 days were obtained from the National Institute of Nutrition, Hyderabad, India and maintained under standard conditions (12 h of light and 12 h of dark schedule, with water and pelleted food ad libitum). Animal procedures employed in the study were cleared by institutional ethical committee.
2.2.1. LH antiserum treatment The antiserum to highly purified ovine LH was raised in adult bonnet monkeys which at a dilution of 1:20 000 bound 75% of iodinated rat LH. The ability of this antiserum to neutralize endogenous LH in rats was established by demonstrating that a single injection of 275 ml of the antiserum to adult male rats (90 days) reduced serum testosterone concentration to 12% of value seen in the control animals that received an equal volume of pre-immune serum (Table 1). Serum testosterone levels were determined by specific radioimmunoassay standardized in the laboratory (Rao and Kotagi, 1989). Immature male rats (19 days old) received 125 ml LH a/s for two days (days 19 and 20) by subcutaneous injection and were sacrificed on day 21 for PLC isolation. Animals which received equal volume of pre-immune monkey serum served as control. 2.3. Isolation of PLC, ILC and ALC Cells were isolated from different age groups of rats, according to the procedure of Klinefelter et al. (1987) and Hardy et al. (1990). Adult Leydig cells were purified by elutriation followed by Percoll density gradient fractionation. In view of the fact there is minimal
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spermatogenic activity in 21 and 35 day old rats only density gradient fractionation of interstitial cells was used for isolation of PLC and ILC. The density gradient was formed by centrifugation at 20 000× g at 4°C for 1 h, PLC were collected between densities of 1.064– 1.070. Similarly, for ILC and ALC fractions between 1.070 and 1.088 were collected. Cells were washed with DMEM-Ham F12, pelleted at 250 × g, and resuspended in DMEM-Ham F12 and used for further studies.
2.4. Cell culture Purified cells (PLC, ILC, ALC) were plated in 96well culture plates (20 000 cells/well in 200 ml DMEMF12 containing 0.1% BSA and 12 mg/ml gentamycin, referred to as culture medium) at 34°C. Cells were allowed to attach for the first 15 h, followed by replacement of the culture medium (200 ml) with or without 100 ng of hCG or 15 ng of IGF-1. Cells were incubated in culture medium containing 10 mM BrdU for 12 h after addition of hCG and 12 h later; cells were processed for determining the incorporation of BrdU by ELISA according to the manufacturer’s instructions.
2.5. Western blotting of PCNA and D-cyclins Leydig cells were homogenized in 50 mM PBS and the solubilised proteins were estimated by Lowry’s method using BSA as standard (Lowry et al., 1951). Fifty mg of the solubilised proteins were subjected to SDS-PAGE (10%) and Western blotting was carried out, according to the procedure of Towbin et al. (1979). Following incubation of the membranes with rat monoclonal antibody to D-cyclins (D1, D2 and D3) or PCNA in 5% aqueous solution of non-fat dry milk powder at a dilution of 1:500, membranes were washed and incubated with rabbit anti-mouse IgG conjugated to horse radish peroxidase. After washing, the membranes were developed to visualize the immunoreactive band using ECL detection system. Intensity of signal was quantitatively measured by densitometry of the films. Table 1 Reduction in serum testosterone levels by LH antiseruma Serum testosterone ng/ml (mean 9SE) Pre-immune 2.72 90.47 serum-treated LH 0.337 90.04 antiserum-treat ed
2.6. Semi-quantitati6e RT-PCR for IGF-1 and IGF-1 receptor Total RNA was isolated from progenitor Leydig cells using TRIzol reagent and intact RNA with A260/280 ratio 1.6 above was used for RT-PCR. One mg of RNA was reverse transcribed using 50 units MMLV-reverse transcriptase with random hexamers in a 20 ml reaction volume for 1 h at 37°C. An aliquot of 2 ml from the RT mixture containing the cDNAs was used for PCR in a 50 ml of reaction volume. The oligonucleotide primers for amplifying IGF-1 were 5%-GCC ACA GCC GGA CCA GAG ACC CTT-3% and 5%-CTA CAT TCT GTA GGT CTT GTT TCC-3% and the oligonucleotide sequence to amplify IGF-1 receptor were 5%-CTG TGT TCT TCT ATG TCC CA-3% and 5%CGA GCT CCC GGT TCA TGG TG-3% (Murphy et al., 1987; Kurachi et al., 1992). Cyclophilin was employed as internal control and primers used to amplify were 5%-GTG GCA AGT CCA TCT ACG-3% and 5%-CAG TGA GAG CAG AGA TTA CAG-3% (Majdic et al., 1996). The expected size of the products were 327 bp, 261 and 380 bp for IGF-1, IGF-1 receptor and cyclophilin, respectively. The PCR was performed within the linear range of amplification to assess their expression levels with cyclophilin as an internal control. The number of cycles performed for IGF-1 and IGF-1 receptor were 30, and 28 for cyclophilin. After an initial denaturation at 94°C for 3 min each cycle included 1 min at 95°C, 1 min at 56°C and 1 min at 72°C except that for IGF-1 receptor the annealing temperature was 52°C. The PCR amplification was carried out for respective number of cycles as stated above and 12 ml of each RT-PCR product was electrophoresed in 2% agarose gel with Ethidium Bromide in 0.045 M Tris–borate/0.001 M EDTA buffer. The difference in intensities of the product following electrophoretic analysis was analysed using EDAS 120 Kodak Gel documentation system.
2.7. Statistical analysis The data were analysed by Kruskal–Wallis ANOVA and a P value less than 0.05 was considered to be statistically significant. The data presented in figures and graphs were representative of at least three independent experiments.
Percentage inhibition (%) – 87.7
LH antiserum (275 ml) was administered to adult male rats (n=6/group) by intraperitoneal route and 24 h later serum testosterone was determined by specific RIA. a
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3. Results
3.1. Purity and functional characteristics of PLC, ILC and ALC The purity of the cells isolated form different age groups of animals was assessed by 3b-hydroxysteroid dehydrogenase histochemical staining PLC and ILC
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Fig. 1. Western blot analysis of cyclin D3 protein in Leydig cells. The homogenates of PLC (lane 1), ILC (lane 2), ALC (lane 3) containing 50 mg of protein were electrophoresed and transferred to nitrocellulose membrane for detection of 30–34 kDa cyclin D3 protein with a mouse monoclonal antibody at a dilution of 1:500 by ECL system (Amersham).
Fig. 2. Western blot analysis for PCNA protein in Leydig cells. The homogenates of PLC (lane 1), ILC (lane 2), ALC (lane 3 represents cells isolated by Percoll gradient fractionation and lane 4 represents cells isolated by elutriation followed by Percoll gradient fractionation) containing 50 mg of protein were electrophoresed and transferred to nitrocellulose membrane for detection of the 36 kDa PCNA protein with a mouse monoclonal antibody at a dilution of 1:500 by ECL system (Amersham).
hCG (100 ng) as monitored by in vitro testosterone production was also assessed. It is to be noted that although the major end product of steroidogenic response by PLC is 5a-androstane-3a,17b-diol and not testosterone as in the case of ALC, still an increase in testosterone can be observed following in vitro incubation of PLC with hCG/LH (ng of testosterone produced/106 cells for 3 h) mean9 SD, n=3 with PLC, control 0.19 0.02, hCG 0.239 0.03 with ALC; control 3.190.1, hCG 25.291.2).
3.2. D-cyclins in Leydig cells The presence of D-cyclins (D1, D2 and D3) were assessed in PLC, ILC and ALC since their association with cyclin-dependent kinase forms an active complex leading to phophorylation of various intracellular substrates resulting in activation of genes involved in DNA synthesis and ultimately enables cells to begin the transition from G1- to S-phase. Western blotting of cyclin D1, D2 and D3 in the samples of purified Leydig cells revealed an absence of cyclin D1 and D2 (data not shown) and presence of cyclin D3, consistent with the known molecular weight 32 kDa. The level of cyclin D3 decreased with increasing age and when the levels of cyclin D3 were expressed relative to PLC, it was observed that while it was 809 3% in the ILC, and was only 3.469 2% in the case of ALC (Fig. 1).
3.3. PCNA in Leydig cells PCNA, a 36 kDa protein is an essential replication factor and hence its immunoreactivity is a useful marker for cell proliferation and its level correlates with other parameters of proliferation. An immunoblot of the Leydig cells proteins, for PCNA revealed an age-dependent reduction in its level as the Leydig cells lose their proliferative capacity and differentiate. When the PCNA levels were expressed relative to PLC it was only 1592% in ILC and 7.49 1.5% in ALC (Fig. 2).
3.4. Effect of hCG on in 6itro Leydig cell proliferati6e capacity
Fig. 3. Effect of addition of hCG on BrdU incorporation in culture in Leydig cells. PLC, ILC and ALC were isolated and cultured as described in Section 2. Bars corresponding to 1, 3, 5 and 2, 4, 6 indicate the BrdU incorporation values without and with hCG. Values represent mean 9SE from three separate experiments cultured in triplicates. Statistical significance tested between groups 2 and 4 and groups 2 and 6 were **P= 0.0082.
were found to be 92% pure and ALC were 90% pure. The response of these cells to a fixed concentration of
The proliferative capacity during the course of postnatal development of Leydig cells with hCG was assessed by DNA synthesis during culture by monitoring BrdU incorporation. Following addition of hCG a significant increase in BrdU incorporation in PLC (0.8 O.D./20 000 cells) was noted. However, it was significantly less in ILC (0.075 O.D./20 000 cells) and completely absent in ALC (Fig. 3). These results indicate that LH stimulates DNA synthesis in PLC and there is a progressive loss in the proliferative capacity of cells isolated from animals of increasing age.
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3.5. Effect of depri6al of endogenous LH on bromodeoxyuridine incorporation, PCNA and cyclin D3
Fig. 4. Effect of in vivo LH deprival on BrdU incorporation by PLCs in culture. PLCs were isolated from Pre-immune serum (PIS) and LH-antiserum (LH a/s) treated groups and cultured as described in Section 2. Values represent mean 9 SE. Data shown from at least three separate experiments (***P=0.0002).
Fig. 5. Western blot analysis of Leydig cell cyclin D3 after LH antiserum treatment. The homogenates of PLC from pre-immune serum-treated (lane 1) and LH antiserum-treated (lane 2) groups containing 50 mg of protein were electrophoresed and transferred to nitrocellulose membrane for detection of 30–34 kDa cyclin D3 protein, with a mouse monoclonal antibody at a dilution of 1:500 by ECL system (Amersham).
In order to ascertain the role of LH in modulation of these regulatory proteins, a passive neutralization study was carried out in immature rats (19 days old), since Leydig cells isolated from this period showed a maximum proliferative capacity. Deprival of endogenous LH for 2 days (days 19 and 20) using a specific antiserum to LH led to total absence of BrdU incorporation when isolated Leydig cells were cultured in the presence of hCG, unlike in the control group in which an 8-fold increase was seen (Fig. 4). When Leydig cells proteins were analyzed by Western blot for cyclin D3 and PCNA the level of cyclin D3 in the LH antiserumtreated group was much smaller when compared with that in the control (Fig. 5). Similarly, the level of PCNA was found to be decreased by 60% in the LH antiserum-treated group (Fig. 6). It has been well-documented that in vitro addition of IGF-1 stimulates DNA synthesis in progenitor Leydig cells (Ge and Hardy, 1997), and our results reveal that when IGF-1 stimulated DNA synthesis was assessed by BrdU incorporation in cells isolated from LH a/s treated rats, there was a significant decrease in incorporation (data not shown). These results indicate that specific deprivation of LH leads to decreased DNA synthesis as assessed by BrdU incorporation and decrease in the levels of cyclin D3 and PCNA proteins which play a key role in G1 to S transition and DNA replication.
3.6. PCR based quantitation of IGF-1 and IGF-1 receptor mRNA le6els Several studies have established an important role for IGF-1 in control of Leydig cell proliferation (Benton et al., 1995). Since our studies revealed an important role for LH and there was loss of IGF-1 and IGF-1 receptor in Leydig cells following deprival of endogenous LH in 21 day old rats. A semi-quantitative RT-PCR analysis revealed a 52.5 9 2.5% reduction in mRNA levels of IGF-1 and 389 2% decrease in IGF-1 receptor (Fig. 7) when expressed relative to cyclophilin. These results suggests that LH is necessary to maintain the mRNA levels for IGF-1 and IGF-1 receptor.
4. Discussion
Fig. 6. Western blot analysis of PCNA in Leydig cells after LH antiserum treatment. The homogenates of PLC from pre-immune serum-treated (lane 1) and LH antiserum-treated (lane 2) groups containing 50 mg of protein were electrophoresed and transferred to nitrocellulose membrane for detection of the 36 kDa protein, with a mouse monoclonal antibody at a dilution of 1:500 by ECL system (Amersham).
Our results demonstrate a decrease in cyclin D3 and PCNA along with a loss in proliferative capacity as a function of age in Leydig cells and also following deprival of endogenous LH in immature rats. In this connection it is pertinent to note that in a recent study it has also been reported that expression of cyclin D3 is strong in endocrine cells secreting steroid hormones and
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Fig. 7. Semi-quantitative RT-PCR for IGF-1 and IGF-1 receptor in progenitor Leydig cells following deprival endogenous LH. RNA isolated purified PLCs were reverse transcribed, cDNA obtained was subjected to a semi-quantitative PCR in the linear range of amplification for (a) IGF-1; (b) IGF-1 receptor and (c) cyclophilin as an internal control. Lanes 1 and 2 correspond to PCR amplified products from pre-immune serum-treated and LH antiserum-treated groups, respectively.
in normal human testes, the cell type-restricted expression patterns were dominated by high levels of cyclin D3 in Leydig cells (Doglioni et al., 1998; Bartkova et al., 1999). Considering this it is appropriate that we have monitored the levels of cyclin D3 as an indicator for proliferative activity of progenitor Leydig cells. The observation that the incorporation of BrdU which is an indicator of DNA synthesis is maximum only in PLC isolated on day 21 when compared with cells isolated on days 35 and 90 suggests that proliferative capacity is maximum in PLC and this age group is the most appropriate to study the role of LH in Leydig cell proliferation. In addition, our results suggest that a sharp decline of cyclin D3 and PCNA in immature and adult Leydig cells could contribute to their loss of proliferative capacity since these proteins mediate the G1 to S transition and DNA synthesis. It is well-known that D-cyclins bind to cyclin-dependent kinase (cdk) 4 or cdk 6 which allows phosphorylation by cdk-activating kinase thereby forming an active complex which phosphorylates retinoblastoma protein RB and other related proteins leading to activation of E2F family of transcription factors. This leads to the activation of numerous genes involved in DNA synthesis and ultimately enables cells to begin the transition from G1- to S-phase (Robker and Richards, 1998). PCNA is a essential replication factor synthesized at the initial stages of G1-phase and has a longer half life accumulat-
ing in the nucleus until mitosis. PCNA binds to DNA polymerase-d and D-cyclins to initiate cell cycle progression (Ogle et al., 1998). Thus immunoreactivity of D-cyclins and PCNA are useful markers of cell proliferation and this correlates with other parameters employed to assess cellular proliferation. The results obtained in the present study reveal that the levels of PCNA and cyclin D3 as assessed by Western blot, are maximum in PLC’s isolated from 21 day old rats and this correlates with their ability to synthesize DNA in vitro following addition of hCG when compared with Leydig cells isolated from day 35 (ILC) and day 90 (ALC). Several factors are known to modulate Leydig cell proliferation but the primary stimulus for the pubertal increase in Leydig cell number has not been established. Of the various factors involved LH has been found to be indispensable for functional differentiation of Leydig cell proliferation is not clear. In contrast to the earlier studies which have employed either impure preparation of Leydig cells or morphometric and histological parameters to assess the role of LH to stimulated Leydig cells proliferation, we have employed purified Leydig cells and also more than one parameter namely, BrdU incorporation, level of PCNA, and cyclin D3 to assess the role of LH in stimulation of Leydig cell proliferation. Before using the Leydig cells isolated from immature rats (21 days) to evaluate the role of LH in Leydig cell proliferation, we have ascertained that this age group is the most appropriate one for proliferation study. In addition, we have employed the procedure standardized and validated by Klinefelter et al. (1987) and Hardy et al. (1990) to isolate Leydig cells and the isolated cells were highly pure as assessed by 3b-HSD staining as well as by the demonstration that cells isolated from different age groups respond to hCG/LH and the maximum response was seen with ALC. Our results of in vitro response studies with PLC to hCG/LH provide additional support to the conclusion that LH stimulates proliferation in these cells in spite of the fact that these cells have low level of LH receptor as reported by Shan and Hardy (1992). It is also known that the circulating levels of LH are low in 18 day old rats and thus it appears that the low level of LH receptor in PLC are adequate to stimulate the proliferative response of the cells to added hCG/LH. However, ALC which have maximum levels of LH receptor do not exhibit proliferative response to added hCG/LH although the ability of LH to induce functional differentiation of ALC is well-documented in literature. Our study also employs the approach of selective neutralization of LH for only two days without the surgical trauma of the hypophysectomy or the other effects likely to be seen following administration of EDS. In addition, the usefulness of specific antibodies to investigate the role of LH in rats and monkeys
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has been well established earlier (Moudgal et al., 1974). Neutralization of LH resulted in total absence of BrdU incorporation by isolated Leydig cells in culture following addition of hCG and a drastic decrease in cyclin D3 and PCNA protein was seen as assessed by immunoblotting. The role of LH in stimulation and maintenance of smooth endoplasmic reticulum which is involved not only in biosynthesis of steroids but also in general protein synthesis (Ewing and Zirkin, 1983) has been well-documented. Long term suppression of LH is associated with Leydig cell atrophy, reduction in steroidogenic enzymes and testosterone synthesizing capacity with drastically diminished smooth endoplasmic reticulum (Wing et al., 1985). Also acute stimulation of Leydig cells with LH involves the synthesis of at least 800 – 900 new proteins (Luo et al., 1998). Hence, deprival of LH for 2 days would have resulted in a loss of cellular integrity due to reduction in cellular proteins ultimately leading to loss in proliferative capacity. It should be noted that while a complete absence of cyclin D3 was noticed, only 60% reduction in PCNA was seen and it could be due to higher half life of PCNA protein (Ogle et al., 1998) when compared with cyclin D3. This indicates that LH is necessary for inducing the changes in the levels of cyclin D3 and PCNA, and regulation of Leydig cell proliferation. Based on the results of an in vitro study, which consisted of monitoring mRNA levels of cyclin A2, cyclin G1 and 3H thymidine incorporation following addition of LH Ge et al. (1996), reported that LH is important for maintenance of PLC proliferation. However, in our present study we have monitored in vitro DNA synthesis, levels of cyclin D3 and PCNA in purified Leydig cells from immature rats deprived of endogenous LH which will reflect the in vivo role of LH in Leydig cell proliferation. Although, results of studies with either transgenic mice overexpressing LH or LH knockout mice are likely to provide additional evidence to establish a role for LH in Leydig cell proliferation, as of now there are no reports of studies with these models. Recent reports on naturally occurring mutations in humans provide evidences for the role of LH in Leydig cell proliferation. Inactivating human LH beta mutation in humans results in absence of Leydig cells (Huhtaniemi et al., 1999) and an inactivating mutation in LH receptor leads to Leydig cell hypoplasia (Laue et al., 1996). However, it is also important to note that knockout involves a permanent loss of a specific gene product. But we have employed passive neutralization which involves selective deprival of LH for a desired period to demonstrate a stage specific function. Results of our studies also reveal that passive neutralization of LH leads to loss of IGF-1 stimulated BrdU incorporation in vitro by downregulating the mRNA levels for IGF-1 and IGF-receptor. The observation that there was a decrease in the BrdU incorpora-
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tion as well as decrease in the mRNA levels of IGF-1 and IGF-1 receptor following LH deprival suggests the possibility that LH can mediate its proliferative effects by regulating IGF-1 and its receptor in Leydig cells. In conclusion, Leydig cells lose their proliferative capacity during their course of post-natal development which is associated with decline in PCNA and cyclin D3 levels. LH plays a major role in Leydig cell proliferation by maintaining the levels of these proteins in a stage specific manner and regulating the IGF-1 network.
Acknowledgements The authors wish to thank Dr M.P. Hardy, Population Council, Center for Biomedical Research, New York for critically reviewing the manuscript and for his valuable suggestions. The authors also wish to thank Prof. A.M. Dharmarajan, University of Western Australia, Perth for reviewing the manuscript and providing the PCNA antibody. Financial assistance from Council of Scientific and Industrial Research, Indian council for Medical Research, Department of Science and Technology, Department of Biotechnology, Government of India and MRC of Canada is greatly acknowledged. Rat LH and hCG used in this study were generously provided by National Hormone and Pituitary Programme, NIDDK, USA.
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