:: Asian Journal of Andrology ::

Studies on LH modulated 8 kDa peptide involved regulation of testosterone production in rat Leydig cells

P. Ramaraj, A. Jagannadha Rao¹

Department of Diabetes, Endocrinology & Metabolism, The University of Hope National Medical Centre, 1500, East Duarte Road, Duarte, CA 91007, USA
¹Department of Biochemistry & Department of Molecular Reproduction, Development & Genetics, Indian Institute of Science, Bangalore-560 0112, India

Asian J Androl 1999 Dec; 1: 191-194

Keywords: Leydig cells; testosterone; regulation; differentiation; steroidogenesis; peptides; LH

Abstract

Aim: To demonstrate the role of the 8 kDa peptide in regulation of testosterone production by rat Leydig cells. Methods: A peptide similar to 8 kDa peptide purified from immature rat Leydig cells was isolated and purified from rat lung cytosol. Immunological and structural similarity between the peptides purified from lung and Leydig cells was established by Western blot and tryptic map comparison respectively. Results: Addition of the 8 kDa peptide 10, 50, 100, and 150 g decreased the production of testosterone in Leydig cells dose-dependently. But the addition of the peptide 150 g along with hCG had no effect on hCG-stimulated increase in testosterone production. Conclusion: In vitro addition of the peptide purified from lung cytosol to adult rat Leydig cells resulted in a concentration-dependent decrease in basal testosterone production although it had no effect on hCG-stimulated testosterone production.

1 Introduction

Developmentally rat Leydig cells undergo three stages maturation designated as progenitor Leydig cells, immature Leydig cells and adult Leydig cells^[1]. Recent studies^[2] have established that these three cell types are distinct in terms of LH receptor content, metabolism of testosterone and ability to respond to LH. The acquisition of steroidogenic machinery and response to LH is complete and maximum once the cells become adult Leydig cells. In an attempt to elucidate the factors involved in regulation of acquisition of response to LH by the immature Leydig cells we have examined the protein profile of the purified Leydig cells of immature and adult rat Leydig cells. These studies revealed the presence of a peptide of approximate molecular weight of 8 kDa in the immature rat Leydig cells which was absent in the adult Leydig cells^[3]. We have also observed an inverse relationship between the ability of the Leydig cells to produce testosterone and the presence of the peptide. In the present report we provide additional evidences which establish the involvement of this peptide in regulation of testosterone production by rat Leydig cells.

2 Materials and methods

2.1 Materials

Adult Wistar rats (90 days old) or immature rats (21 days old) which were maintained under 14-h light and 10-h darkness of schedule were maintained in groups of 2 or 3 in polypropylene cages. Water and food were made available ad libitum. Details of procedures employed to isolate purified Leydig cells from immature and adult rats have been described earlier^[4]. Our earlier studies have revealed that the level of the 8 kDa peptide was modulated only in the Leydig cells by LH, although a significant quantity of the peptide was present in other tissues such as heart, kidney, liver and brain besides lung which had maximum quantity. Since the objective of the present study was to demonstrate the role of the 8 kDa peptide in regulation of testosterone production by rat Leydig cells, it was necessary to obtain the peptide in sufficient quantity. However, Leydig cells have relatively low concentration of the 8 kDa peptide and since lung was found to be a rich source of the peptide, the peptide was purified from rat lung. Differential centrifugation analysis of lung and Leydig cell homogenate revealed that the peptide was localized in the cytosol (data not shown). Accordingly the peptide was purified by subjecting the lung cytosol to preparative SDS PAGE using rat Leydig cell peptide as a reference standard. The gel was stained with potassium chloride and the band corresponding to 8 kDa peptide was cut out and eluted. The eluted peptide was checked for homogenity by electrophoresis. The identity between the peptides isolated from lung cytosol and immature rat Leydig cytosol was ascertained by comparing the two-dimensional tryptic map of the two peptides. Tryptic map analysis was carried out as described earlier^[5].

2.2 Effect of addition of 8 kDa peptide from rat lung on testosterone production by the rat Leydig cells

Purified Leydig cells (50 000 cells in 200 L) from adult rats were incubated in the presence or absence of 160 pg of highly purified hCG to assess the response of the cells. Purified Leydig cells were also incubated along with hCG (160 pg) and 150 g of 8 kDa peptide isolated from the rat lung. In addition, different quantities of the 8 kDa peptide isolated from rat lung were added to Leydig cells in the absence of hCG at 34. Following incubation for 3 h, medium and cells were separated by centrifugation at 500g and medium was analyzed for testosterone by specific RIA as described earlier^[6]. The specificity of the effect of addition of 8 kDa peptide was ascertained by addition of rat serum albumin (RSA) (150 g) instead of the 8 kDa peptide as well as preincubation of the 8 kDa (100 g) peptide at 37 for 1 h before addition to the Leydig cells with gamma globulin isolated from rabbit antiserum (500 L) to 8 kDa peptide or equal quantity of normal rabbit serum.

3 Results

3.1 Immunological and structural similarity between the peptides purified from lung and Leydig cells

It can be seen from the results presented in Figure 1 and 2 that the peptide isolated from the rat lung cytosol is homogenous and exhibits the same mobility as seen in the case of purified peptide from rat Leydig cells and it cross-reacts with antiserum to 8 kDa peptide isolated from immature rat Leydig cells. Comparison of the tryptic maps of 8 kDa peptides isolated from lung cytosol and rat Leydig cells revealed that the pattern of peptides was very similar, indicating that the peptide isolated from lung cytosol is very similar to the 8 kDa peptide isolated from the rat Leydig cells (data not shown). This observation validates the experiments using peptide isolated from lung since sufficient quantity of purified peptide could not be isolated from immature rat Leydig cells in view of the low yield.

Figure 1. SDS-PAGE analysis of crude cytosol from rat lung, purified peptide from lung cytosol and purified 8 kDa peptide from immature rat Leydig cells.
Lane 1: 250 g of protein from rat lung cytosol.
Lane 2: 50 g of purified peptide from rat lung cytosol.
Lane 3: 100 g of purified peptide from immature rat Leydig cells.
Lane 4: Cytochrome C (50 g).
Figure 2. Western blot analysis of 8 kDa peptide purified from rat lung cytosol with antiserum to 8 kDa peptide from rat Leydig cells. Antiserum was used at a dilution of 1:1000.
Lane 1: 50 g of 8 kDa peptide from rat Leydig cell cytosol.
Lane 2 & 3: 50 & 100 g of 8 kDa peptide from lung cytosol.

3.2 Effect of addition of 8 kDa peptide isolated from rat lung on testosterone production by adult rat Leydig cells

It can be seen from the results presented in Figure 3 that the rat Leydig cells respond to added hCG as seen by the increased testosterone (Lane 2) over the control (Lane 1). Addition of the peptide along with hCG had no effect on hCG-stimulated increase in testosterone production (Lane 3). However, following addition of the peptide alone to the Leydig cells, there was a significant dose-dependent decrease in the quantity of testosterone produced and the effect was maximum with the addition of 100 g of peptide (Lane 4-7) in the absence of the hCG. In contrast, addition of 150 g of RSA had no effect on testosterone production (Lane 8) and preincubation of the peptide with its specific antiserum abolished the inhibitory effect (Lane 10) while preincubation of the peptide with normal rabbit serum retained the inhibitory effect (Lane 9). Interestingly addition of 150 g of protein from cytosolic extract from the rat adrenal gland had no effect on the testosterone production (Lane 11).

Figure 3. Effect of in vitro addition of 8 kDa peptide on basal testosterone production by adult rat Leydig cells. Each bar represents means of 3 values.
Lane 1: Unincubated control.
Lane 2: 160 pg hCG.
Lane 3: 160 pg hCG+150 g 8 kDa peptide.
Lane 4-7: 10, 50, 100 & 150 g peptide alone.
Lane 8: 150 g RSA.
Lane 9: 150 g peptide preincubated with 500 L antiserum to 8 kDa peptide.
Lane 10: 150 g peptide preincubated with 500 L normal rabbit serum.
Lane 11: 150 g of protein from adrenal cytosol.

4 Discussion

Elucidation of the factors involved in regulation of acquisition of steroidogenic function by rat Leydig cells has been the subject of intense investigation^[7,8]. While LH has been demonstrated to be indispensable for maintenance of steroidogenic function of Leydig cells in the adult rats, its role in regulation of growth of Leydig cells in the neonatal and immature rats has not been well defined^[9,10]. It has been suggested that during this period LH may not have an important role since LH receptor content of the Leydig cells is very low during the neonatal stage and also deprival of LH by use of specific antiserum to LH capable of neutralizing endogenous LH did not have any drastic effect on testis weight or steroidogenesis.

Our earlier studies have demonstrated the presence of 8 kDa peptide in the immature rat Leydig cells which exhibited an inverse relationship with the testosterone producing capacity^[3]. The quantity of the peptide decreased with increase in age and also with the acquisition of the ability by the Leydig cells to produce testosterone. Interestingly the quantity of the peptide decreased following administration of hCG, which stimulated testosterone production by the Leydig cells. This was found to be true even with the immature female rats which were treated with hCG which is known to stimulate steroidogenesis. In view of the problems associated with obtaining sufficient quantity of purified peptide from immature rat Leydig cells, we have employed the rat lung as a source to obtain sufficient quantity of the peptide. Comparison of tryptic maps of the peptides from rat Leydig cells and lung revealed that they were very similar and hence could be used in place of the rat peptide. In the present study we have observed that following in vitro addition of the 8 kDa peptide purified from rat lung cytosol to adult rat Leydig cells, there is a concentration-dependent decrease in basal (incubated) testosterone production, compared to the unincubated controls. However, it was not effective in inhibiting the hCG-stimulated testosterone production and in the presence of hCG, even the basal production of testosterone was unaffected indicating that hCG is able to override the inhibitory effect of the peptide. This suggests that the action of the peptide is unlikely to be at the level of binding of hCG to its receptor. It is pertinent to note that we have observed earlier that this peptide is not part of the LH receptor^[4]. Considering these facts and the observation that only basal production of testosterone is inhibited it is possible that this peptide interferes at a step prior to hCG binding possibly such as transport of cholesterol. The specificity of the effect of peptide was ascertained by addition of the peptide, which was incubated with specific antiserum which resulted in lack of inhibition, while the peptide, which was incubated with normal rabbit serum, retained its inhibitory activity. Another supportive evidence which suggests its involvement in regulation of steroidogenesis in Leydig cell is the fact that addition of 150 g of protein from cytosol of adrenal of an adult male rat which is steroidogenically very active was not effective in inhibiting testosterone production.

Stimulation of specific protein synthesis in the adrenal following treatment of rats with ACTH or by LH in the rat Leydig cells has been reported earlier^[11,12]. However, ours is the first report of a presence of peptide which is negatively modulated by LH in the rat Leydig cells and as suggested earlier^[4]this peptide may be part of an overall mechanism in deciding whether cholesterol should be channeled into steroid hormone production or not.

5 Acknowledgments

The authors wish to acknowledge the financial assistance received from Indian Council of Medical Research (ICMR) and Council of Scientific Industrial Research (CSIR) government of India. AJR is thankful to NIDDK, National Hormone and Pituitary Programme, USA for generous gift of hormones used in the study.

References

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[8] Hardy MP, Kelce WR, Klinefelter GR, Ewing LL. Differentiation of Leydig cell precursors in vitro: a role of androgen. Endocrinol 1990; 127: 488-90.
[9] Christensen AK, Peacock KC. Increase in Leydig cell number in testes of adult rats treated chronally with an excess of hCG. Biol Reprod 1989; 22: 383-91.
[10] Moore A, Findlay K, Morris ID. In vitro DNA synthesis in Leydig and other interstitial cells of the rat testes. J Endocrinol 1992; 134: 247-55.
[11] Janszen FH, Brain AC, Van Driel JA, Van Der Molen HJ. Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells. Biochem J 1978; 170: 9-15.
[12] Dazord A, Gallet D, Saez JM. Effect of corticotropin treatment in vivo on the synthesis of a specific adrenal cytosolic protein. Biochem J 1978; 176: 233-9.

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Correspondence to A. Jagannadha Rao, Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, INDIA.
E-mail: ajrao@biochem.iisc.ernet.in
Tel. +91-80-309 2308; 309 2548 Fax: +91-80-334 5999
Received 1999-09-03 Accepted 1999-12-02