Does calcitonin gene-related peptide act as a chemoattractant for rat gubernacular cells?

Does Calcitonin

Gene-Related Peptide Act as a Chemoattractant Rat Gubernacular Cells?

By Yoshifumi Sugita, Georgia

A. Pax-ton, Suzanne Hasthorpe, and John

for

M. Hutson

Melbourne, Australia l Calcitonin gene-related peptide KGRP) receptors are located in the cremaster muscle of the gubernaculum of rats, and causes gubernacular contraction in vitro, suggesting that CGRP plays an important role in testicular descent. It has been postulated that CGRP released from the genitofemoral nerve acts as a “chemoattractant” for gubernacular migration to the scrotum. The aim of this study is to investigate whether the gubernacular cells of rats exhibit a chemotactic response to CGRP in vitro. Chemotaxis of gubernacular cells from Sprague-Dawley rats (I to 6 days old) was measured using blind-well chambers. The migration of cells, which passed from the upper to the lower compartment through a polycarbonate filter, were counted using microscopy. The lower compartment included lo-“, lo-lo, 1O-g, and lo-* M CGRP or phosphate buffered saline (PBS) as control. The chemotactic index (Cl) was defined as the ratio of migration toward CGRP versus PBS as control. There was no significant migration even at varying CGRP concentrations. Isolated rat gubernacular cells therefore did not exhibit a chemotactic response to CGRP and the role CGRP plays in testicular descent still remains unclear. This result does not exclude the possibility that the gubernaculum responds to CGRP as a whole organ rather than as individual cells. Cop yrighf o 1997 by W. B. Saunders Company INDEX WORDS: Testicular descent, chemotaxis, gene-related peptide (CGRP), rat gubernaculum.

calcitonin

T

HERE ARE MANY controversies surrounding the mechanism of normal testicular descent. One hypothesis is the biphasic model of testicular descent.’ In this hypothesis, the first stage is transabdominal descent in which the testis moves from its initial position on the posterior abdominal wall to the inguinal region. The second stage, known as inguinoscrotal descent, involves migration of the testis to the scrotum. Subsequently, the genitofemoral nerve (GIN) hypothesis was postulated to explain inguinoscrotal testicular descent.2 It was proposed that calcitonin gene-related peptide (CGRP) released from GFN controlled the second stage descent of the testis.3 This hypothesis is supported by the finding that CGRP receptors are located in the cremaster muscle of the rat gubernaculum.” Moreover, contractions of rodent gubemacula are observed in vitro following CGRP injection5s6 However, the mechanism by which CGRP appears to control gubemaculuar migration remains unclear. CGRP is a known chemoattractant for F9 embryonal carcinoma cells,’ human T lymphocytes8 and airway epithelial cells.9 Similarly CGRP could act as a chemoattractant for rat gubemacular cells. The testis and epididymis may descend to the scrotum in conjunction with the Journal

offediatric

Surgery,

Vol32,

No 1 (January),

1997: pp 15-17

gubemaculum along a concentration gradient of CGRP released by the GIN. The aim of this study is to investigate whether the gubemacular cells of rats exhibit a chemotactic response to CGRP in vitro. MATERIALS

AND

METHODS

Male Sprague-Dawley rats maintained at the Animal Research Laboratory in the Royal Children’s Hospital were used for all experiments. These rats were kept in controlled conditions with a fixed temperature and a 12-hour light/dark cycle. Rats (n = 164) were killed at days 1 through 6 and a lower abdominal skin incision was made. The gubemaculum was removed transperitoneally by cutting its proximal and distal attachment to the epididymis and inguinal region, respectively, using an operating microscope. For testing of chemotaxis on freshly isolated cells, the gubemacula were placed together into the Iscove’s medium (Gibco, Grand Island, NY) and digested in 0.1% protease (Sigma Chemical Co, Castle Hills, NSW) with Iscove’s medium containing 5% fetal calf serum (FCS) (Trace Biosciences Ltd, Grand Island, NY) for 30 minutes at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Following this procedure, Iscove’s medium with 10% FCS was added to the cells and centrifuged at 1,000 ‘pm, then suspended and allowed to incubate at room temperature for 30 minutes. The solution was washed twice with Iscove’s medium and resuspended in Iscove’s medium with 1% bovine serum albumin (BSA). The cell number was counted using Trypan blue exclusion and a hemocytometer and adjusted to a concentration of lo6 cells per mL for use in the chemotaxis assay. For chemotactic analysis of gubemacular cells established m culture the gubemacula were minced to obtain a single cell suspension and placed mto a 25 cm* culture flask with Iscove’s medium containing 20% FCS. After either 1 week or 1 month culture (medium changed weekly), the cells were prepared for chemotactic assay by addttton of 5 mL of 0.5% Trypsin (Sigma Chemical Co) to harvest the cells, and then they were incubated at 37“C for 3 minutes. The medium was aspirated and placed into a tube contammg 1 mL of Iscove’s contaimng 20% FCS. The solution was centrifuged at 1,200 rpm for 5 minutes at 4°C and washed once with Iscove’s medium containing 1% BSA. The cell number was totalled by Trypan blue exclusion and adjusted to 10h cells/ml for chemotaxis assay. Chemotactic assays were carried out m Boydeni” and blind-well chambers.” Preliminary studies with both chambers showed that blind-well chambers gave more reproducible results, and therefore these were used for the quantnative study described here. Polycarbonate filters, 7 to 10 pm thickness with pore size 8 lmr, polyvmyl-pyrrohdme (PVP) free (Nuclepore, Pleasanton, CA)” were coated with 0 1% gelatine. Soluttons of human CGRP

From the E Douglas Stephens Surgrcal Research Laboratory Royal Children’s Hospital, Melbourne. Australra. Presented at the 29th Annual Meeting of the Pactjic Association of Pediatric Surgeons, Singapore, May 12-15, 1996. Address reprint requests to Professor John M. Hutson, Department of General Surgery, Royal Children S Hospital, Flemington Rd, Parkville, Mctoria 3052, Australia. Copyright 0 1997 by WB. Saunders Cornpan) OOZZ-3468/97/3201-0004$03.00/O

15

16

SUGITA

(10-u, lo-lo, 10M9, lo-*) (AMRAD Corporation, Melbourne) andPBS were individually prepared by adding Iscove’s medium. Aliquots of 185 pL of medium, which contained either 18.5 pL of CGRP or PBS were placed in the lower chamber of the blind well. Then 200 uL of gubernacular single-cell suspension was added to the upper chamber. All chambers were incubated for 4 hours at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After incubation, the upper solution was aspirated and the filters stained with Hematoxylin. Determination of the degree of chemotaxis was carried out as described elsewhere.t2 The cells adherent to the undersurface of the filter and were counted as migrating cells in 10 high-power fields (magnification X400). Experiments were conducted with different concentrations of CGRP, using phosphate-buffered saline as control. The chemotactic index was defined as the ratio of migration toward CGRP versus saline as control. Student’s unpaired t test was used to compare groups. For freshly isolated cells, each concentration of CGRP was analyzed in three replicate chambers, and for cells established in culture, 13 chambers were used at each concentration.

ET AL

12 p = 0.89

10 8 z

I

I p = 0.85

8

I

I T p = 0.15

.o 2 5 E Q)

I 64-

5 2-

RESULTS

Initially freshly prepared (uncultured) cells of the gubernacula were used. The chemotactic index in each group (three replicates) was less than two and there was no significant difference between the various concentrations of CGRP (Fig 1). The gubernaculum consists mainly of epithelial cells, cremaster muscle cells, and mesenchymal cells. These results showed that none of these cells individually exhibited a chemotactic response to CGRP Next we used tissue culture of gubernacular cells, which largely consists of mesenchymal fibroblasts. No difference in chemotaxis was observed after 1 week or 1 month of culture (results not shown). Neither group of cultured cells showed a chemotactic response to CGRP, and there was no significant difference between the different CGRP concentrations (13 replicates) (Fig 2).

8

I

3!2

k .-0 c:

I

p=

I

p = 0.24 1

0.36 I

Concentration

of CGRP (M)

Fig 1. Chemotactic index using freshly isolated gubernacular cells fn = 3 chambers per group). The mean and standard error was calculated in each group. Student’s t test was used to compare chemotaxis at different CGRP concentrations.

Fig 2. Chemotactic response using cultured gubernacular cells In = 13 chambers per group). The mean and standard error was calculated in each group. Student’s t test was used to compare chemotaxis at different CGRP concentrations.

DISCUSSION

Calcitonin gene-related peptide (CGRP) is a 37 amino acid derived from alternate splicing of the initial RNA transcript of the calcitonin gene.13It is distributed throughout the central and peripheral nervous system and has various functions. CGRP-like immunoreactivity is observed in the spinal motor nucleus of the GFN.’ CGRP also acts as a chemoattractant for F9 embryonal carcinoma cells7 human T cell lymphocytes,* and human airway epithelial cells.9 The mechanism of normal testicular descent still remains unclear. Hutson has proposed a two-stage model for testicular descent. During the first stage of descent, known as transabdominal descent, the testis is anchored to the inguinal region by enlargement of the gubemaculum. Inguinoscrotal descent occurs between 26 and 40 weeks’ gestation3 and involves active gubemacular migration, because it has been shown that prior to this phase the gubemaculum ends at the abdominal wall and is not found in the future scrotum.14 The GFN hypothesis was later proposed2 to explain inguinoscrotal descent. This hypothesis proposes that androgens released from the developing testis act primarily on the GFN via the central nervous system; the GFN then acts to stimulate gubemacular migration. Subsequently, CGRP has been identified as the neurotransmitter present in both the GFN nucleus3 and GFN branches of the rodent,r5 and is known to cause rhythmic contractions of the whole gubemaculum. The mechanism by which CGRP may control gubernacular migration remains unknown. One suggestion is that CGRP may cause gubemacular migration by a process of chemotaxis. l6 The experiments performed in

CGRP

CHEMOTAXIS

IN RAT GUBERNACULAR

CELLS

17

this study aimed to determine whether CGRP is chemotactic for individual rodent gubemacular cells in vitro. The gubemaculum consists of the epithelium (peritoneum), cremaster muscle, and mesenchyme. First, by using freshly isolated gubernacular cells, we tested whether any of thesecell types could exhibit a chemotactic responseto CGRP Cultured gubemacular cells, which had a predominantly fibroblastic appearancewere also testedfor a chemotactic response to CGRP Lung fibmblasts have been shown previously not to exhibit a chemotactic response to CGRP.” Our experiments show that CGRP does not act as a chemoattractant for any components of the gubemaculum.

Despite lack of CGRP-induced chemotaxis of isolated gubemacular cells, the role of the gubemaculum remains central to the process of testicular migration to the scrotum,14 as exogenous CGRP injected ectopically diverts gubemacular migration in the flutamide-treated rat with cryptorchidismr7 and intrascrotal CGRP 8-37, an antagonist of CGRP, causes a delay in testicular descent in rnice.18 Although we have found that CGRP does not have a chemotactic effect on isolated gubemacular cells, further investigation will be needed to understand the role CGRP plays in migration of the intact gubemaculum.

REFERENCES 1. Hutson JM: A biphasic model for the hormonal control of testicular descent. Lancet 24:419-421, 1985 2. Beasley SW, Hutson JM: Effect of division of genitofemoral nerve on testicular descent in the rat. Aust NZ Surg 57:49-51, 1987 3. Larkins SL, Hutson JM, Williams MPL: Localization of calcitonin gene-related peptide immunoreactivity within the spinal nucleus of the genitofenoral nerve. Pediatr Surg Int 6:176-179, 1991 4. Yamanaka J, Metcalfe SA, Hutson JM: Demonstration of calcitonin gene-related peptide receptors in the gubemaculum by computerhzed densitometry. J Pediatr Surg 26:621-623, 1991 5. Momose Y, Griffiths AL, Hutson JM: Testicular descent III. The neonatal gubemaculum shows rhythmic contraction in organ culture in response to calcitonin gene-related pepode. Endocrinology 131:28812884,1992 6. Shono T, Goh DW, Momose Y, et al: Physiological of calcitoni gene-related peptide (CGRP) on gubemacular with or without denervation. J Pediatr Surg 30591-595, 7. Gerbaud P, Segond N, Moukhtar MM, et al: calcitonin gene-related peptide are chemotactic for carcinoma cells. Endocrinology 129:2530-2534, 1991 8. Foster CA, Mandak B, Kromer E, et al: Calcitonin peptide is chemotactic for human T lymphocytes. Ann

effects in vitro contractility 1995 Calcitonin and F9 embryonal gene-related N Y Acad Sci

657:397-404,1992 9. Shoji

S, Ertl RF. Linder

I, et al: Bronchial

epithelial

cells respond

to insulin and insulin-like growth factor-l as a chemoattractant. Am J Respir Cell Mol Biol2:553-557, 1990 10. Boyden S: Chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. J Exp Med 115:453-466, 1962 11. Wilkinson PC: Micropore filter methods for leucocyte chemotaxis. Methods Enzymol 162:38-50, 1988 12. Coates TD: An integrated system for quantitatton of chemotaxis using a 48-well millipore filter assay. Comp Meth Prog Biomed 38:177-192, 1992 13. Rosenfeld MG, Mermod JJ, Amara SG, et al: Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNAprocessing. Nature 304: 129-135, 1983 14. Heyns CF: The gubemaculum during testicular descent in the human fetus. JAnat 153:93-112, 1987 15. Larkins SL, Hutson JM: Fluorescent antegrade labelling of the genitofemoral nerve shows that it supplies the scrotal region before migration of the gubemaculum. Pediatr Surg Int 6:167-171. 1991 16. Hutson JM, Terada M, Zhou B, et al: Normal testicular descent and the aetiology of cryptorchidism. Adv in Anat Embryo1 Cell Biol 132:1-51, 1995 17. Abe T, Hutson JM: Calcitonin gene-related peptide injected ectopically alters gubemacular migration in the Butamide-treated rat with cryptorchidism. Pediatr Surg Int 9:551-554, 1994 18. Samarakkody UKS, Hutson JM: Intrascrotal CGRP 8-37 causes a delay in testicular descent in mice. J Pediatr Surg 27:874-875, 1992