Cell membrane and mitotic markers show that the neonatal rat gubernaculum grows in a similar way to an embryonic limb bud

doi:10.1016/j.jpedsurg.2007.04.035

Journal of Pediatric Surgery

Volume 42, Issue 9, September 2007, Pages 1566-1573

https://doi.org/10.1016/j.jpedsurg.2007.04.035 Get rights and content

Abstract

Aim/Background

How the gubernaculum guides the testis into the scrotum remains controversial, with various proposals from passive inversion to active growth. We aimed to determine if the gubernaculum contains an area of active proliferation, such as a “progress zone” in a growing embryonic limb bud, using a fluorescent cell membrane marker, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate [DiIC₁₂(3)], to trace cell migration, and 5-bromodeoxyuridine (BUDR) (a thymidine analogue) as a mitotic marker.

Methods

Gubernacula were collected from neonatal male rats (n = 42, day 1-2, Sprague-Dawley) and cultured with calcitonin gene–related peptide (CGRP; 714 nmol/L). 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate–coated glass beads (diameter, 150-212 μm) were placed next to the bulb for the first 3 hours. Gubernacula were cultured for 3, 18, and 24 hours, then frozen sections cut and examined by confocal microscopy (wavelength, 549 nm). In a second experiment, pups not exposed to exogenous CGRP (n = 53, day 0, Sprague-Dawley) were injected intraperitoneally with BUDR (50 mg/kg of body weight); gubernacula were collected at 2, 48, 72, and 96 hours postinjection (PI), sectioned, and stained using immunohistochemistry to count the number of BUDR-positive cells per 100 cells (labeling index) in the bulb, cremaster, cord, and epididymis.

Results

After 24 hours' culture with CGRP, the bulb showed an oval region (diameter, 300 μm) of high fluorescence, and the cremaster region showed elongated cells migrating out of the bulb. When cultured without CGRP, the same oval region contained no fluorescence. In vivo BUDR labeling index increased in all areas until 48 hours postinjection and then decreased most rapidly in the bulb (P < .05), in the presence of endogenous CGRP from the genitofemoral nerve.

Conclusions

The rat gubernaculum contains a putative progress zone, such as in a growing limb bud, in the presence of CGRP. Cells migrate out of this zone to form cremaster muscle. We hypothesize that proliferation in the bulb elongates the gubernaculum, whereas proliferation of cremaster cells would increase gubernacular diameter. This brings to “life” the gubernaculum as an actively growing organ in contrast to the inert ligament connecting the testis to the scrotum portrayed in most anatomy textbooks.

Section snippets

Animals

Neonatal Sprague-Dawley male rats (n = 95) were obtained from the Royal Children's Hospital (Melbourne, Australia) and the University of Melbourne (Melbourne, Australia) animal research laboratories. All rats were maintained in normal shoebox rat cages and fed commercial pellets and water ad libitum. They were kept in a controlled environment with a 12-hour light/dark cycle. This project was approved by the Murdoch Childrens Research Institute (Melbourne, Australia) animal experimentation

1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate study in vitro

The gubernaculum in culture took up the DiIC₁₂(3) by diffusion. The fluorescent labeling pattern remained relatively constant after 3 and 18 hours of culture (Fig. 2). However, after 24 hours of culture, the gubernaculum cultured with CGRP displayed an oval-shaped region of fluorescent labeling in the bulb. The peripheral future cremaster region contained distinctly labeled elongated cells. In comparison, after 24 hours of culture without CGRP, an almost inverted image of a dark oval-shaped

Discussion

Using a lipophilic dye that labels intact cell membranes of live cells, we visualized a zone of active growth in response to CGRP in the gubernacular bulb. Dye uptake was uniform throughout the bulb for up to 18 hours of culture. After 24hours, however, the labeling pattern was significantly different in the presence of CGRP, which has been shown to enhance proliferation in the bulb [8]. The fluorescent elongated cells interspersed in the lateral part of the gubernaculum are likely to have

References (19)

J.M. Hutson
Testicular feminization: a model for testicular descent in mice and men
J Pediatr Surg
(1986)
N.S. Shenker et al.
A new role for androgen in testicular descent: permitting gubernacular cell proliferation in response to the neuropeptide, calcitonin gene–related peptide
J Pediatr Surg
(2006)
J.S. Elder et al.
Androgenic sensitivity of the gubernaculum testis: evidence for hormonal/mechanical interactions in testicular descent
J Urol
(1982)
M. Terada et al.
Ontogeny of gubernacular contraction and effect of calcitonin gene–related peptide in the mouse
J Pediatr Surg
(1994)
M. Terada et al.
Characterization of the gubernacular contractile response to calcitonin gene–related peptide in the mouse
J Pediatr Surg
(1995)
S.K. Lam et al.
Does the gubernaculum migrateduring inguinoscrotal testicular descent in the rat?
Anat Rec
(1998)
J. Rajfer et al.
Testicular descent
Birth Defects Orig Artic Ser
(1977)
N. Shubin et al.
Fossils, genes and the evolution of animal limbs
Nature
(1997)
D. Summerbell
Interaction between the proximo-distal and antero-posterior co-ordinates of positional value during the specification of positional information in the early development of the chick limb-bud
J Embryol Exp Morphol
(1974)

There are more references available in the full text version of this article.

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2023, Human Reproductive and Prenatal Genetics
Male reproductive development is a complex process requiring precise endocrine and genetic signals to regulate morphological changes. In this chapter, we review the genetic cues that induce male sexual differentiation from the bipotential gonad and genital tubercle to the development of the testes and penis, respectively. To begin, SRY production in XY gonads initiates the male pathway there by inducing testicular development and Mullerian regression. The newly formed testes must then descend into the scrotum to later produce functional sperm. Testicular descent is a biphasic process consisting of INSL3-dependent transabdominal descent and androgen-dependent inguinoscrotal descent. Many genes involved in testicular development are also integral in the development of the external genitalia. The external genitalia develops from a sexually undetermined genital tubercle that undergoes hormone-driven sexually dimorphic development. Finally, we examine birth defects that can arise from perturbations in the intricate system that regulates normal male reproductive development.
Developmental genetics of the male reproductive system
2018, Human Reproductive and Prenatal Genetics
Male reproductive development is a complex process requiring precise endocrine and genetic signals to regulate morphological changes. In this chapter, we review the genetic cues that induce male sexual differentiation from the bipotential gonad to the development of the testes and penis. To begin, SRY production from the male gonads induces testicular development and Mullerian regression. The newly formed testes must then descend into the scrotum to produce functional sperm. Testicular descent is a biphasic process consisting of INSL3-dependent transabdominal descent and androgen-dependent inguinoscrotal descent. Many genes involved in testicular development are also integral in the development of the external genitalia. The external genitalia develops from a gender neutral genital tubercle that undergoes hormone-driven differentiation. Finally, we examine birth defects that can arise from perturbations in the intricate system that regulates normal male reproductive development.
The syndrome of Spigelian hernia and cryptorchidism: A review of paediatric literature
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Citation Excerpt :
In rodent models, specialised peritoneum covering the urogenital ridge grows into the gubernaculum, forming the processus vaginalis, which precedes the testis in the migrating testis-gubernaculum complex, completing formation of the inguinal canal [43,47,48]. Androgen-mediated ‘programming’ of the genitofemoral nerve (GFN) allows secretion of calcitonin gene related peptide (CGRP) from the GFN, which controls gubernacular migration out of the inguinal canal to the scrotum, with growth characteristics similar to an embryonic limb bud [37,48–54]. The absence of CGRP has multiple deleterious effects on the gubernaculum, including apoptosis in the gubernacular growth centre [55] and abnormal gubernacular migration [54,56].
The purpose of this study is to present a summary of paediatric Spigelian hernia (SH) reported to date, and discuss possible aetiology of SH associated with ipsilateral ectopic testis (SH-ET).
Search of PubMed, Medline, Embase, and CINAHL was performed using keywords “Spigelian hernia”. The following were extracted from articles describing paediatric SH: demographics, site and contents of SH, comorbidities, proposed aetiology, presence of ipsilateral inguinal canal (IC) and gubernaculum (G).
There were 78 patients with 88 hernias (69 male, 19 female), including 55 male (19 left, 22 right, 7 bilateral) and 16 female (5 left, 5 right, 3 bilateral) nontraumatic SHs. In nontraumatic male SH, 29 hernias contained testis (10 left, 11 right, 4 bilateral), 15 did not, 10 had no data. Of 29 SH-EH, 15 were lacking IC and G, 3 were missing IC (no G data) and 2 had absent G (no IC data).
The combination of SH and cryptorchidism is increasingly recognised as a distinct syndrome. However, there is controversy as to the pathogenic mechanism of this association. We hypothesise SH-ET develops because the G, and therefore IC and line of descent, become cranially ‘mislocated’ along the mammary line, which overlies the Spigelian fascia.
SH is rare in children. SH-ET may result by testicular descent to an ectopic site along the embryological mammary line.
Mathematical modelling of gubernaculum during inguino-scrotal migration shows limb bud characteristics
2013, Journal of Pediatric Surgery
Citation Excerpt :
Recent studies show that the gubernaculum in rodents has many molecular properties that are similar to an embryonic limb bud. For example, the tip of the gubernaculum can be labelled with a lipophilic vital dye to reveal the undifferentiated mesenchymal bulb that behaves like a limb bud growth centre, or “progress zone” [3]. Indeed, the gubernacular tip of the neonatal rat can be excised and grafted just like a limb bud progress zone, consistent with it containing a growth centre [2].
The gubernaculum is postulated to grow like an embryonic limb bud during inguinoscrotal descent in rodents. Recently, modelling of limb bud growth suggests the undifferentiated, distal “progress zone" provides molecular morphogenic signals, rather than cell division, as previously thought. We aimed to develop a mathematical gubernacular growth model, hypothesising that it would mimic limb buds through evolutionary conservation.
Histology was done on Sprague–Dawley rats (day 2, 8; n = 7/group) to determine gubernacular length, width, cell density in distal growth centre, middle and proximal cremaster muscle. Analysis of measurements enabled gubernacular growth modelling under variable growth centre sizes/densities, assuming no apoptosis.
Modelling found that gubernacular growth occurred mostly within cremaster muscle, rather than primarily in the undifferentiated mesenchymal tip, despite its higher mitotic rate. The growth centre accounted for ≤ 10% of total gubernacular enlargement/elongation.
These results suggest the gubernaculum elongates by proliferation throughout cremaster muscle like a limb bud. The distal undifferentiated tip may provide signalling molecules for growth, which could be a fruitful source for causes of failed migration/elongation in cryptorchidism.
Cryptorchidism and endocrine disrupting chemicals
2012, Molecular and Cellular Endocrinology
Citation Excerpt :
In rodents, androgens have been suggested to affect the gubernacular migration and growth via or together with genitofemoral nerve and its neurotransmitter calcitonin gene-related peptide (CGRP) (Beasley and Hutson, 1987; Ng et al., 2005; Park and Hutson, 1991; Shenker et al., 2006), but the role of the genitofemoral nerve and CGRP in humans is unclear (Hutson et al., 2010). It has been hypothesized that during migration the gubernaculum has similar properties as a developing embryonic limb bud (Huynh et al., 2007; Na et al., 2007; Nightingale et al., 2008). In addition to androgens, also INSL3/RXFP2 signalling have been suggested to have a role in the inguinoscrotal descent of the testis (Bay and Andersson, 2011; Yuan et al., 2010).
Prospective clinical studies have suggested that the rate of congenital cryptorchidism has increased since the 1950s. It has been hypothesized that this may be related to environmental factors. Testicular descent occurs in two phases controlled by Leydig cell-derived hormones insulin-like peptide 3 (INSL3) and testosterone. Disorders in fetal androgen production/action or suppression of Insl3 are mechanisms causing cryptorchidism in rodents. In humans, prenatal exposure to potent estrogen diethylstilbestrol (DES) has been associated with increased risk of cryptorchidism. In addition, epidemiological studies have suggested that exposure to pesticides may also be associated with cryptorchidism. Some case–control studies analyzing environmental chemical levels in maternal breast milk samples have reported associations between cryptorchidism and chemical levels. Furthermore, it has been suggested that exposure levels of some chemicals may be associated with infant reproductive hormone levels.
The common fetal development of the mammary fat pad and gubernaculum
2011, Journal of Pediatric Surgery
Citation Excerpt :
The lack of a correlation between AR expression and proliferation suggests that rapid growth is less important at this time than differentiation to permit subsequent remodeling. The differential expression of activated Notch-1 and Jagged-1 throughout the E19 gubernaculum suggests that Notch-1 signaling may regulate differential morphology in the gubernaculum, maintaining undifferentiated cells in the tip, promoting growth characteristics such as the progress zone of an embryonic limb bud [23], and regulating AR function in the more caudal gubernaculum at this time, an established role of Notch-1 [24]. Further study of this pathway is warranted to elucidate this putative action more fully.
Recent work both from our laboratory and in marsupial models of testicular descent suggests a strong connection between the mammary line and gubernacular migration. This study investigated the relationship between the mammary fat pad (MFP) that underlies the mammary line and the developing gubernaculum by fluorescent immunohistochemistry.
Rats at E17 and E19 were fixed and processed for immunohistochemistry. Sagittal sections of male fetuses were stained with antibodies against androgen receptor (AR), prolyl-4 hydroxylase β, Desmin, activated Notch-1, Jagged-1, and Ki-67. These were analyzed by fluorescent confocal microscopy.
At E17 and E19, the MFP anlage forms a continuous distribution of fibroblasts passing immediately adjacent the gubernaculum to the future scrotum. Within this exists a distinct subpopulation of fibroblasts expressing AR distributed over the path of inguinoscrotal descent of the gubernaculum. Proliferation and Notch-1 signaling were similar throughout the MFP with differential Notch-1 signaling in the E19 gubernaculum.
This investigation has identified the presence of a distinct AR-expressing subpopulation of MFP fibroblasts over the path of inguinoscrotal descent during the key androgenic programming window of this phase. This unique developmental pattern is consistent with a prime role for the MFP in testicular descent.

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View full text

Cell membrane and mitotic markers show that the neonatal rat gubernaculum grows in a similar way to an embryonic limb bud

Abstract

Aim/Background

Methods

Results

Conclusions

Section snippets

Animals

1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate study in vitro

Discussion

J Pediatr Surg

J Pediatr Surg

J Urol

J Pediatr Surg

J Pediatr Surg

Does the gubernaculum migrateduring inguinoscrotal testicular descent in the rat?

Anat Rec

Testicular descent

Birth Defects Orig Artic Ser

Fossils, genes and the evolution of animal limbs

Nature

Interaction between the proximo-distal and antero-posterior co-ordinates of positional value during the specification of positional information in the early development of the chick limb-bud

J Embryol Exp Morphol