Calcitonin gene-related peptide is a survival factor, inhibiting apoptosis in neonatal rat gubernaculum in vitro.
BACKGROUND
Testicular descent is proposed to occur in 2 stages. During the second stage, calcitonin gene-related peptide (CGRP) released from the genitofemoral nerve (GFN) causes maximal mitosis in the gubernacular bulb. As normal development requires a balance between cell proliferation and apoptosis, this study explored the effect of CGRP on apoptosis in the rat gubernacular bulb.


METHODS
Gubernacula were collected from male Sprague-Dawley rats at birth (D0) or 2 days post birth (D2), and placed in organ culture for 24 hours with or without CGRP (0.001 mol/L). The D2 rats were pretreated with capsaicin (sensory nerve toxin) or flutamide (antiandrogen) or untreated. D0 rats were untreated (n = 64). Sections of the bulb were stained using the TUNEL method to identify apoptotic cells. Apoptosis was calculated as the percentage of positive cells per hundred cells.


RESULTS
Normal Sprague-Dawley rat gubernacula showed reduced apoptosis when cultured with CGRP, in D0 (7.0% vs 4.8%, P < .05) and D2 (4.9% vs 2.3%, P < .001). Greater apoptosis occurred at D0 compared to D2, without CGRP added (7.0% vs 4.9%, P < .05) and with CGRP (4.8% vs 2.3%, P < .001). For D2 gubernacula, capsaicin treatment increased apoptosis compared to controls, without CGRP added (4.9% vs 7.3%, P < .05) and with CGRP (2.3% vs 6.7%, P < .001). There was no difference in apoptosis when cultured with or without CGRP (7.3% vs 6.7%, nonsignificant) after capsaicin treatment. Flutamide treatment increased apoptosis compared to controls, but only with CGRP (2.3% vs 7.3%, P < .001). There was no difference in apoptosis when cultured with or without CGRP (7.1% vs 7.3%, nonsignificant) after flutamide.


CONCLUSIONS
Calcitonin gene-related peptide (CGRP) acts as a survival factor in the rat gubernaculum, possibly to steer cells away from a defined apoptotic pathway. Greater apoptosis occurs earlier in development. However, in vivo CGRP released from the genitofemoral nerve may be required to prevent apoptosis, as shown by pretreatment with the sensory nerve toxin capsaicin. Androgen is also involved in the pathway controlling apoptosis, as androgen blockade with flutamide inhibited the action of CGRP.
Chan
Jessica J
JJ
Douglas Stephens Surgical Research Laboratory, Murdoch Childrens Research Institute, Melbourne, Victoria 3052, Australia.
Farmer
Pam J
PJ
Southwell
Bridget R
BR
Sourial
Magdy
M
Hutson
John M
JM
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology*; D002211:Capsaicin; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D005485:Flutamide; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D008297:Male; D008938:Mitosis / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018709:Statistics, Nonparametric; D013737:Testis / Q000187:drug effects* / Q000254:growth & development*
D005485:Flutamide; D015740:Calcitonin Gene-Related Peptide; D002211:Capsaicin