Demonstration of calcitonin gene-related peptide receptors in the gubernaculum by computerized densitometry

doi:10.1016/0022-3468(92)90389-O

Journal of Pediatric Surgery

Volume 27, Issue 7, July 1992, Pages 876-878

https://doi.org/10.1016/0022-3468(92)90389-O Get rights and content

Abstract

The gubernaculum appears to guide inguinoscrotal testicular descent by migration into the scrotum ahead of the testis. Calcitonin gene-related peptide (CGRP) has been found in the scrotal branches of the genitofemoral nerve of neonatal rats, and is known to stimulate gubernacular motility in vitro. This study aimed to identify CGRP receptors in the gubernaculum, which should be present if CGRP mediates gubernacular migration toward the scrotum. Gubernacular sections from neonatal male rats were incubated with [¹²⁵I]-human CGRP as well as a variety of unlabeled neuropeptides. By using computerized densitometry, the quantitation of CGRP binding derived from in vitro autoradiography demonstrated a distinctive distribution of binding sites for CGRP over the developing cremasteric muscle in the gubernaculum. The binding analysis showed a single class of sites with a dissociation constant (Kd) of 2.13 nmol/L and a receptor density of 27.4 fmol/mg polymer. These results endorse the hypothesis that CGRP released from the nerve acts directly on the cremaster via its own receptors, which have not been described previously.

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Cited by (35)

Testicular descent: A review of a complex, multistaged process to identify potential hidden causes of UDT
2022, Journal of Pediatric Surgery
Citation Excerpt :
CGRP is a 37-amino acid peptide that is generated by alternative splicing of the calcitonin gene mRNA. In rodent gubernaculum, there are CGRP receptors in the cremaster muscle [75] and CGRP causes contractility of the cremaster in organ culture while also stimulating mitosis and supressing apoptosis [76]. This peptide is likely to provide a key directional signal in the migration of the testis to the scrotum [73].
What causes normal descent of the testis in a fetus, and what goes wrong with this complex process to cause undescended testes (UDT), or cryptorchidism? Over the last 2 decades, most authors searching for the cause(s) of UDT have looked at the 2 main hormones involved, insulin-like hormone 3 (Insl3) and testosterone (T)/ dihydrotestosterone (DHT), and their known upstream (hypothalamic-pituitary axis) and intracellular ‘downstream’ pathways. Despite these detailed searches, the genetic causes of UDT remain elusive, which suggest the aetiology is multifactorial, and/or we are looking in the wrong place.
In this review we highlight the intricate morphological steps involved in testicular descent, which we propose may contain the currently ‘idiopathic’ causes of UDT. By integrating decades of research, we have underlined many areas that have been overlooked in the search for causes of UDT.
It is quite likely that the common causes of UDT are still hidden in these areas, and we suggest examining these processes is worthwhile in the hope of finding the common genetic anomalies that lead to cryptorchidism. Given the fact that a fibrous barrier preventing descent is often described at orchidopexy, examination of the extracellular matrix enzymes needed to allow gubernacular migration may be a fruitful place to start.
This review of the complex anatomical steps and hormonal regulation of testicular descent highlights many areas of morphology and signalling pathways that have been overlooked in the search for causes of UDT.
Undescended Testes and Testicular Tumors
2010, Ashcraft's Pediatric Surgery
UNDESCENDED TESTES AND TESTICULAR TUMORS
2009, Ashcraft's Pediatric Surgery, Fifth Edition
Calcitonin gene-related peptide stimulates mitosis in the tip of the rat gubernaculum in vitro and provides the chemotactic signals to control gubernacular migration during testicular descent
2008, Journal of Pediatric Surgery
Citation Excerpt :
It could be caused by the developing cremaster muscle contracting to deviate the tip or directional growth of the tip by cells present at the tip responding by mitosis to CGRP. Calcitonin gene–related peptide binding sites have been reported in the cremaster muscle in 2-day-old rats [16]. The tip of the gubernaculum, which is known to contain undifferentiated mesenchymal cells, has been found in vivo to actively proliferate [3], supporting the results here.
We investigated whether calcitonin gene–related peptide (CGRP) released from sensory genitofemoral nerve branches could stimulate rodent gubernacular growth and provide chemotactic signals for directing inguinoscrotal gubernaculum migration in vitro.
Neonatal rat gubernacula containing a developing cremaster sac (n = 60) were removed at days 0, 2, 4, 6, 8, and 10 (n = 10 per age; n = 5 per experimental group) and placed in organ culture for 24 hours with or without added CGRP (720 nmol/L). The gubernacula were stained for bromodeoxyuridine (BrdU) immunohistochemistry. Cells were counted (3 × 100 cells) in the mesenchymal tip of the gubernaculum to find the percentage of BrdU uptake. A further group of neonatal rat gubernacula (n = 21 per group) were placed in organ culture on an agar platform with 5 agarose beads soaked in either PBS or 10⁻⁶ mol/L CGRP placed approximately 0.8 to 1 mm on each side of the tip of the cremaster sac. After 72 hours, the position of the gubernaculum was compared with its starting position and any deviation measured.
Exogenous CGRP caused a significant increase in BrdU uptake in the tip of the gubernaculum in 0-day-old rats compared with control cultures. Two-way analysis of variance in the cellular proliferation pattern between gubernacula cultured ± CGRP between 0 and 10 days showed a significant difference (P < .001). The cultures containing CGRP-impregnated beads caused significant (P < .01) deviation of the tip of the gubernaculum toward the beads, whereas the controls demonstrated no net movement of the tip.
These studies demonstrate that mitosis in the tip of the rat gubernaculum is significantly increased in response to CGRP in vitro. Also, CGRP may provide chemotactic signals to control inguinoscrotal gubernacular migration in the rat.
The anatomy of the cremaster muscle during inguinoscrotal testicular descent in the rat
2007, Journal of Pediatric Surgery
Citation Excerpt :
Husmann and Levy [6] proposed that CGRP has no role in human testicular descent as it only acts on the developing cremaster in rats, although the muscle is a small component of the human gubernaculum. Certainly, CGRP receptors have only been found in the rat cremaster [18], although at that time the GB was not examined. However, in this study, capsaicin caused rapid disappearance of the bulb, whereas excess CGRP (as in the TS rat) caused persistence of the mesenchymal core of the bulb.
Extrapolation of rat testicular descent studies to humans has been criticized because of anatomical differences of the cremaster muscle. Human cremaster is described as a thin strip rather than a large, complete sac as in rats, which is proposed to be more important in propelling the testis during descent. This study investigated cremaster muscle anatomy and ontogeny in both normal and cryptorchid rat models.
Gubernacula from 4 groups of neonatal rats were sectioned longitudinally and transversely: normal Sprague-Dawley, capsaicin pretreated, flutamide pretreated, and congenital cryptorchid rats. Gubernacula were stained with hematoxylin-eosin, Masson trichrome, and desmin immunohistochemistry to study muscle development.
Myoblasts are more numerous at the gubernacular tip, whereas the most differentiated muscle is proximal. Rat cremaster develops as an elongated strip rather than a complete sac derived from abdominal wall muscles. Flutamide and capsaicin pretreatment disrupts development.
Rat cremaster muscle develops as a strip, bearing close resemblance to human cremaster muscle, permitting extrapolation of cremaster function to human testicular descent. The cremaster muscle appears to differentiate from the gubernacular tip during elongation to the scrotum, and requires intact sensory innervation and androgen.
The role of sympathetic innervation in the developing rat gubernaculum
2007, Journal of Pediatric Surgery
Citation Excerpt :
There is a dose response to CGRP in organ culture [7,8], and the gubernaculum contains binding sites for CGRP over the developing cremaster muscle [19,20]. The source of CGRP has been shown to be sensory nerve fibres in the genitofemoral nerve, which acts independently of acetylcholine [18]. Apart from stimulating contractility [21], CGRP has recently been shown to cause cell division within the gubernaculum [22,23].
Testicular descent occurs in several steps, but the mechanism remains unknown. Recently, sympathetic nerves have been proposed to have a role. This study aimed to see if adrenergic agonists and antagonists affected the neonatal rat gubernacular cremaster sac in organ culture.
Cremaster sacs were collected from 2-day-old Sprague-Dawley male rats (n = 90) and placed in organ culture with/without (1) calcium chloride (0.45-1.8 mmol/L), (2) rat calcitonin gene-related peptide (CGRP) (714 nmol/L), (3) nifedipine (0.1-100 nmol/L), (4) isopreneline (10 μmol/L), and (5) guanethidine (10 μmol/L). Gubernacula were observed over 2 days for rhythmic peristalsis (>120 beats per minute for >1 minute).
Addition of CGRP stimulated rhythmic contractility but only in the presence of calcium, with a dose-response to the level of calcium ions. Contractions induced by CGRP with calcium could be obliterated in a dose-response by nifedipine. Isoprenaline caused some increase in contractions with calcium but less than that seen with CGRP. No augmentation of effect was seen with CGRP and isopreneline together, and the level of contractility was the same with guanethidine.
Calcitonin gene-related peptide is the main effector for contractility of the rat cremaster sac, as long as calcium ions are present. Adrenergic agonists and antagonists had limited effects. Contractility could be inhibited with nifedipine, consistent with CGRP acting via a calcium-dependent pathway.

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Supported in part by a Clinical Research Scholarship from the Royal Children's Hospital Research Foundation, Melbourne, Australia.

Presented at the 38th Annual International Congress of the British Association of Paediatric Surgeons, Budapest, Hungary, July 24–26, 1991.

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Demonstration of calcitonin gene-related peptide receptors in the gubernaculum by computerized densitometry☆

Abstract

J Urol

J Steroid Biochem

J Pediatr Surg

Life Sci

Anal Biochem

Partial characterization of the androgen receptor of the newborn rat gubernaculum

Biol Reprod