Inguinoscrotal Testicular Descent Research Articles

The SPARC-related modular calcium binding 1 (Smoc1) regulated by androgen is required for mouse gubernaculum development and testicular descent.

Testicular descent occurs in two consecutive stages: the transabdominal stage and the inguinoscrotal stage. Androgens play a crucial role in the second stage by influencing the development of the gubernaculum, a structure that pulls the testis into the scrotum. However, the mechanisms of androgen actions underlying many of the processes associated with gubernaculum development have not been fully elucidated. To identify the androgen-regulated genes, we conducted large-scale gene expression analyses on the gubernaculum harvested from luteinizing hormone/choriogonadotropin receptor knockout (Lhcgr KO) mice, an animal model of inguinoscrotal testis maldescent resulting from androgen deficiency. We found that the expression of secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 (Smoc1) was the most severely suppressed at both the transcript and protein levels, while its expression was the most dramatically induced by testosterone administration in the gubernacula of Lhcgr KO mice. The upregulation of Smoc1 expression by testosterone was curtailed by the addition of an androgen receptor antagonist, flutamide. In addition, in vitro studies demonstrated that SMOC1 modestly but significantly promoted the proliferation of gubernacular cells. In the cultures of myogenic differentiation medium, both testosterone and SMOC1 enhanced the expression of myogenic regulatory factors such as paired box 7 (Pax7) and myogenic factor 5 (Myf5). After short-interfering RNA-mediated knocking down of Smoc1, the expression of Pax7 and Myf5 diminished, and testosterone alone did not recover, but additional SMOC1 did. These observations indicate that SMOC1 is pivotal in mediating androgen action to regulate gubernaculum development during inguinoscrotal testicular descent.

BDNF is upregulated by androgen in the inguinal fat pad of immature mice and may regulate inguinoscrotal testicular descent.

Androgens control rodent inguinoscrotal testicular descent during a "programming window" (E12-17). It is proposed that androgen masculinises the genitofemoral nerve, but the mechanism remains unknown. We investigate androgen receptor (AR)-containing target organs: inguinal fat pad (IFP) and mammary bud (MB), supplied by the genitofemoral nerve, hypothesizing that neurotrophic factors may retrogradely masculinise the GFN. The IFP, MB and bulbocavernosus (BC) muscle were collected at E12.5/E17.5 from androgen receptor knockout (ARKO) mice and wild-type (WT) littermates. Immunofluorescence and gene expression (RT-qPCR; n = 8/group) for Bdnf, active (TrkB) and inactive (truncated TrkB) receptors, Cntf and Cntf receptor were performed. In the IFP at E12.5, ARKO TrkB mRNA expression was significantly downregulated compared to WT males (p < 0.0026). By E17.5, there was increased Bdnf expression (p < 0.0233). The MB had no differences at E12.5 and had regressed in WT males by E17.5. The BC had no differences at E12.5, but at E17.5 had significant upregulation of Bdnf expression in ARKO, compared to WT males. There were no differences in CNTF or CNTF receptor expression. Androgen alters active TrkB and Bdnf expression in the IFP. IFP Bdnf signalling may regulate "masculinisation" of the GFN sensory nerves to indirectly control inguinoscrotal testicular descent. Androgen mediates neurotrophin release in the inguinal fat pad in mice, which may facilitate normal testicular descent by masculinising the GFN by peripheral uptake of neurotrophin. This is the first study to examine the role of neurotrophins in testicular descent. This suggests novel steps in the mechanical process of normal testicular descent that may be abnormal in some children with undescended testes.

Neurotrophin signaling in a genitofemoral nerve target organ during testicular descent in mice

Background/AimIt has been proposed that androgens control inguinoscrotal testicular descent via release of calcitonin gene-related peptide (CGRP) from a masculinised genitofemoral nerve (GFN). As there are androgen receptors in the inguinoscrotal fat pad (IFP) during the window of androgen sensitivity (E14-17 in mouse embryos), we tested the hypothesis that neurotrophins in the IFP may masculinise the sensory fibers of the GFN supplying the gubernaculum and IFP prior to gubernacular migration. MethodsAndrogen-receptor knockout (ARKO) and wild-type (WT) mouse embryos were collected at E17, with ethical approval (AEC 734). Sagittal sections of IFP, mammary area and bulbocavernosus (BC) muscle were processed for standard histology and fluorescent immunohistochemistry for ciliary neurotrophic factor (CNTF), ciliary neurotrophic factor receptor (CNTFR) and cell nuclei (DAPI). ResultsIn the ARKO mouse CNTFR immunoreactivity (CNTFR-IR) was increased in the IFP but decreased in BC. Perinuclear staining of CNTF-IR was seen in mouse sciatic nerve but only weakly in IFP. In the mammary area, also supplied by GFN, there were no differences in IR staining. ConclusionThis study found CNTFR-IR in the IFP was negatively regulated by androgen, suggesting that CNTF signaling may be suppressed in GFN sensory nerves to enable CGRP expression for regulating gubernacular migration in the male, but not the female. The indirect action of androgen via the GFN required for testicular descent may be one of the sites of anomalies in the putative multifactorial cause of cryptorchidism.

Regression of the Mammary Branch of the Genitofemoral Nerve May be Necessary for Testicular Descent in Rats

Inguinoscrotal testicular descent has been proposed to occur via sensory fibers of the sexually dimorphic genitofemoral nerve, which release a neurotransmitter, calcitonin gene related peptide, to guide the migrating gubernaculum into the scrotum. We hypothesize that androgen mediated regression of the genitofemoral nerve mammary branch is necessary for inguinoscrotal descent in rats. We compared the spatiotemporal development of the genitofemoral nerve in control and antiandrogen treated rats. A total of 29 Sprague-Dawley® rats were collected (animal ethics committee approval A644) in control and antiandrogen treated groups (flutamide, embryonic days 16 to 19, 75 mg/kg body weight/5% ethanol + oil) on embryonic days 17 and 19, and on postnatal day 2. Sagittal sections of the gubernaculum and its surrounding structures were processed for standard histology and immunohistochemistry for androgen receptor, nerves (Tuj1), calcitonin gene related peptide (marker for genitofemoral nerve) and cell nuclei (DAPI). The inguinal mammary bud, its adjacent androgen receptor and genitofemoral nerve mammary branch (containing calcitonin gene related peptide) persisted from embryonic day 17 to postnatal day 2 in all antiandrogen treated males, yet regressed in all control males by postnatal day 2. Antiandrogens resulted in the persistence of the mammary branch and inguinal mammary bud. Persistent genitofemoral nerve mammary branches may arrest or slow down gubernacular migration by releasing calcitonin gene related peptide in the mammary inguinal fat pad, thus reducing the chemotactic gradient to calcitonin gene related peptide from genitofemoral nerve branches in the distal scrotum. We hypothesize that this process may be related to antiandrogen induced cryptorchidism in the rodent.

The effect of flutamide on expression of androgen and estrogen receptors in the gubernaculum and surrounding structures during testicular descent

Background/Purpose Inguinoscrotal testicular descent is controlled by androgens between embryonic days E16-19, but androgen receptor (AR) and estrogen receptor (ER) locations are unknown. We aimed to find AR, ER α, and ER β in the gubernaculum and inguinal fat pad (IFP) in normal rats and after flutamide treatment. Methods Sprague-Dawley timed-mated rats were injected with flutamide (75 mg/kg body weight/5% ethanol + oil) on E16-19 or vehicle alone. Male fetuses or pups (5-10/group) were collected at E16; E19; and postnatal (P) days 0, 2, 4, 8. Sections were prepared for hematoxylin and eosin or immunohistochemistry for AR, ERα, and ERβ. Receptor labeling was quantitated as distinct nuclear labeling/100 μm 2 in gubernaculum and IFP. Results There was minimal gubernacular AR-labeling until E19, dramatically increasing postnatally. By contrast, at E16-E19 there was significant IFP AR immunoreactivity suppressed by flutamide ( P < .05). No ERα expression was observed, but ERβ was expressed in both gubernaculum and IFP, maximally at E16, but unchanged by flutamide. Conclusions During the androgen sensitivity window (E16-19), the gubernaculum contains ERβ but minimal ERα or AR, while the IFP, which is supplied by the genitofemoral nerve, contains abundant AR that are flutamide-sensitive. These results suggest that the IFP could be the site of androgenic action controlling gubernacular development.

Cremaster Muscle Myogenesis in the Tip of the Rat Gubernaculum Supports Active Gubernacular Elongation During Inguinoscrotal Testicular Descent

Cryptorchidism is a common abnormality and normal testicular descent is controlled by the gubernaculum. The cremaster may originate from abdominal muscles during gubernacular eversion or alternatively it may develop inside the gubernaculum. We studied cremaster myogenesis to determine how it develops. Coronal sections of the pelvis were prepared from male Sprague-Dawley® rats and from males treated prenatally with the antiandrogen flutamide at embryonic day 19, and postnatal days 10, 19 and 35 after receiving ethical approval. Immunohistochemical stains were prepared for Ki67, Pax-7, myogenin, myosin heavy chain 7, Myh1, Myh2, Myh4, embryonic myosin, and slow and cardiac troponin T. Cell counts of the 1) gubernacular tip, 2) proximal gubernaculum/cremaster muscle and 3) adjacent abdominal wall are shown as a percent of positive fibers or positive cells per area. Throughout embryonic day 19, and postnatal days 10 and 19 proliferation (Ki67) was maximal at the gubernacular tip (p <0.001), as were muscle stem cells markers (Pax-7 p <0.05), early myogenesis (myogenin p <0.001) and immature muscle (Myh7, and slow and cardiac troponin T p <0.0001). In contrast, secondary (fast twitch, Myh1, 2 and 4) fibers were more common in abdominal muscles (p <0.0001). Differences in muscle maturity and composition decreased with time. Flutamide treated rats showed more cellular proliferation than controls postnatally on postnatal day 10 (p <0.001) as well as persistent immature embryonic myosin at the tip from postnatal day 19 (p <0.05). Results show that the rat cremaster muscle is more immature at the gubernacular tip, consistent with myogenesis occurring in the gubernaculum during migration to the scrotum, as proposed in humans.

Development of the Gubernaculum During Testicular Descent in the Rat

Gubernacular elongation during inguinoscrotal testicular descent and cremaster muscle development remains poorly described in mammals. The role of the genitofemoral nerve (GFN) remains elusive. We performed detailed histological analysis of testicular descent in normal rats to provide a comprehensive anatomical description for molecular studies. Fetuses and neonatal male offspring (5-10 per group) from time-mated Sprague-Dawley dams (embryonic days 15, 16, and 19; postnatal days 0, 2, and 8) were prepared for histology. Immunohistochemistry was performed for nerves (Class III tubulin, Tuj1) and muscle (desmin). At embryonic days 15 and 16, the gubernaculum and breast bud are adjacent and both supplied by the GFN. By embryonic day 19, the breast bud has regressed and the gubernacular swelling reaction is completed. Postnatally, the gubernacular core regresses, except for a cranial proliferative zone. The cremaster is continuous with internal oblique and transversus abdominis. By postnatal day 2 (P2), the gubernaculum has everted, locating the proliferative zone caudally and the residual mesenchymal core externally. Eversion creates the processus vaginalis, with the everted gubernaculum loose in subcutaneous tissue but still remote from the scrotum. By P8, the gubernaculum has nearly reached the scrotum with fibrous connections attaching the gubernaculum to the scrotal skin. A direct link between GFN, gubernaculum, and breast bud suggests that the latter may be involved in gubernacular development. Second, the cremaster muscle is continuous with abdominal wall muscles, but most of its growth occurs in the distal gubernacular tip. Finally, gubernacular eversion at birth brings the cranial proliferative zone to the external distal tip, enabling gubernacular elongation similar to a limb bud.

Hidden in plain sight: the mammary line in males may be the missing link regulating inguinoscrotal testicular descent

Background/Purpose Inguinoscrotal testicular descent is controlled by androgens and the genitofemoral nerve, but the trigger for what makes the gubernaculum become a migratory organ like a limb bud remains unknown. Recent observations in the flutamide-treated rat suggested a link with the mammary line. We aimed, therefore, to reassess histologic anatomy in 2 different rodent models of androgen blockade, the testicular feminisation mouse (TFM) and the flutamide-treated rat. Methods Neonatal TFM mice and fetal and neonatal rats after pretreatment of dams with an antiandrogen, flutamide (75 mg/kg; sunflower oil; days 16-19), were prepared for histologic analysis of the inguinal region and compared with fetal and neonatal controls. Results Fetal control rats (E15.5 days) showed a mammary bud just outside the future inguinal canal adjacent to the gubernaculum. Neonatal TFM mice showed persistence of the inguinal breast bud supplied by the genitofemoral nerve. Flutamide-treated rats (D2) showed the gubernaculum surrounded by a persisting breast bud. Conclusions The inguinal mammary line is adjacent to the gubernaculum in fetal rodents, and after androgen blockade, the gubernaculum becomes connected to the breast. The male mammary line, which is hidden in plain sight outside the inguinal canal, is made visible by androgen blockade. It may be the missing link in testicular descent, regulating gubernacular migration.

The antiandrogen flutamide perturbs inguinoscrotal testicular descent in the rat and suggests a link with mammary development

Aim Inadequate androgen activity is a likely cause of cryptorchidism in humans, affecting inguinoscrotal testicular descent. Flutamide, a nonsteroidal antiandrogen, produces cryptorchidism in rats. We aimed to determine the anatomical and histologic effects of flutamide. Methods Time-mated Sprague-Dawley female rats were injected subcutaneously with flutamide (75 mg/kg in sunflower oil) on days 16 to 19 of pregnancy. Embryonic (E) and postnatal (P) male offspring were collected (E16, E19, P0, P2, P4, P8) in control and flutamide-treated groups (n = 5-10). Samples were fixed in 4% paraformaldehyde. Five-micrometer-thick sections were prepared for hematoxylin and eosin, trichrome and immunohistochemical stains (Desmin, TuJ1, Ki67). This identified muscle and neural cells and areas of cell proliferation. Results Postnatally, the gubernaculum in flutamide-treated rats had more mesenchyme and muscle than controls. Gubernacular eversion failed, and mammary tissue persisted around the gubernaculum in flutamide-treated rats. Flutamide had no effect on embryonic gubernacular anatomy and histology. Conclusions Prenatal androgens altered postnatal gubernacular anatomy and histology in the postnatal period. Our findings indicate that the failure of gubernacular differentiation and migration may be because of the ongoing presence of mammary tissue in the region of the external inguinal ring.

INSL3/RXFP2 Signaling in Testicular Descent

Mutations of the insulin-like peptide 3 (INSL3) hormone or its receptor, RXFP2, cause intraabdominal cryptorchidism in male mice. Specific RXFP2 expression in mouse gubernacula was detected at embryonic day 14.5 and markedly increased after birth in the developing cremaster muscle, as well as in the epididymis and testicular Leydig and germ cells. INSL3 treatment stimulated cell proliferation of embryonic gubernacular and Leydig cells, implicating active INSL3-mediated signaling. The transcription factor SOX9, a known male sex determination factor, upregulated the activity of the RXFP2 promoter. INSL3 is sufficient to direct the first transabdominal phase of testicular descent in the absence of hypothalamic-pituitary-gonadal axis signaling or Hoxa10, although these factors are important for inguinoscrotal testicular descent. Similarly, conditional ablation of the androgen receptor gene in gubernacular cells resulted in disruption of inguinoscrotal descent. We performed mutation screening of INSL3 and RXFP2 in human patients with cryptorchidism and control subjects from different populations in Europe and the USA. Several missense mutations were described in both the INSL3 and RXFP2 genes. A novel V39G INSL3 mutation in a patient with cryptorchidism was identified; however, the functional analysis of the mutant peptide did not reveal compromised function. In more than 2000 patients and controls analyzed to date, the T222P RXFP2 mutation is the only one strongly associated with the mutant phenotype. The T222P mutant receptor, when transfected into 293T cells, had severely decreased cell membrane expression, providing the basis for the functional deficiency of this mutation.

The anatomy of the cremaster muscle during inguinoscrotal testicular descent in the rat

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.

Development and descent of the testis in relation to cryptorchidism

The testis descends in two phases. Animal studies suggest, that the transabdominal descent of the testis depends on the insulin-like hormone 3 (INSL3). Androgens are important in the inguinoscrotal testicular descent in animals and humans. In general, the cause of cryptorchidism is unknown and the aetiology is possibly multifactorial. Histological changes in cryptorchid testes demonstrate disturbed development. Since testicular descent is regulated by testis-derived hormones, cryptorchidism may reflect a functional defect of the testis.

Over Expression of Insulin-Like 3 Does Not Prevent Cryptorchidism in GNRHR or HOXA10 Deficient Mice

Insulin-like 3 hormone is critical for the induction of growth and differentiation of gubernacular ligaments during embryonic testicular descent. Mice with mutation of insulin-like 3 or its receptor show high intra-abdominal cryptorchidism. We tested whether transgenic over expression of Insl3 can reverse inguinoscrotal cryptorchidism in mice deficient in Gnrhr or Hoxa10 genes. Hoxa10 and Gnrhr deficient mice were intercrossed with Insl3 transgenic mice. The phenotype of the mutant mice and expression of the genes involved in testicular descent were analyzed. Using quantitative reverse transcriptase-polymerase chain reaction we evaluated expression of the genes in neonatal gubernacular cells on INSL3 (Phoenix Pharmaceuticals, Belmont, California) and testosterone stimulation. Transgenic over expression of Insl3 failed to restore normal testicular descent in Hoxa10 or Gnrhr deficient males. Histological evaluation did not reveal any differences in Insl3 transgenic gubernacula in either mutant. In mutant females Insl3 over expression resulted in transabdominal descent of the ovaries to the low abdominal position with the subsequent development of inguinal hernia. Expression of androgen receptor, insulin-like 3 receptor and Hoxa10 was not affected after incubation of neonatal gubernacular cells with insulin-like 3 or androgen. The results suggest that insulin-like 3 is sufficient to direct the first transabdominal phase of testicular descent in the absence of hypothalamic-pituitary-gonadal axis signaling or Hoxa10 but their presence is important for inguinoscrotal testicular descent.

The effect of a prenatal androgen disruptor, vinclozolin, on gubernacular migration and testicular descent in rats

Background/purpose Androgen has been shown to regulate inguinoscrotal testicular descent. This study aims to clarify the effect of one of the major endocrine disrupters, vinclozolin (V), on both gubernacular migration and inguinoscrotal testicular descent in rats. Methods Time-pregnant rats were segregated into 2 groups. In group I, the rats were administered 200 mg/kg/d of V by gavage on days 15 to 18 of gestation. In group II, the rats were administered the same volume of solvent and were used as controls. At birth, the anogenital distance was measured in pups, and gubernacular migration was examined at 10 days of age in some of male offspring. Next, the incidence of testicular descent and the growth of external genitalia were investigated in the remaining male offspring at 60 days of age. The χ 2 test was used for statistical analysis of the results. Results At birth, the anogenital distance (AGD) index decreased significantly more in group I than in group II in male offspring. However, there was no significant difference in the AGD index between the 2 groups in the female offspring. At 10 days of age, an aberrant migration of the gubernaculum was found in the 51.5% of V-treated rats in group I. At 60 days of age, the incidence of cryptorchidism was 57.7% in group I and 0% in group II ( P < .05). In addition, hypospadias with cleft phallus and pseudo vagina with a blind pouch also were observed in some of the V-treated rats. Conclusions Prenatal administration with V thus caused intrauterine defects, which resulted in testicular maldescent caused by the induction of an aberrant migration of the gubernaculum associated with an abnormal extension of the processus vaginalis, and this may have been caused by the antiandrogenic effect of V in utero.

Dose-dependent effect of phthalate ester on testicular descent in pre-and post natal rats.

Mono-n-butyl phthalate (MBP) was administered to pregnant rats from the 15th to the 17th gestational day to investigate the dose-dependent effect of phthalate ester on testicular descent in both pre- and postnatal rats. Thirty pregnant rats (280-330 g) were separated into five groups and administered 1.0 g/kg/day MBP in group I, 0.5 g/kg/day in group II, 0.25 g/kg/day in group III, 0.125 g/kg/day in group IV from the day 15 to 17 of gestation. The rats in group V were only administered solvent and acted as controls. The position of the testis was evaluated in both pre- and postnatal rats. This was significantly higher in 20-day old fetuses in groups I and II than in groups III, IV or V. After birth, the incidence of undescended testis was also higher in groups I and II compared with those of groups III, IV or V. The undescended testes were located in the abdominal cavity in groups I and II, whereas they were located at the inguinal region in group III. The lowest dose that induced an adverse effect on testicular descent was 0.25 g/kg/day. In conclusion, maternal MBP inhibited testicular descent in prenatal rats and thus dose-dependently induced undescended testes postnatally. High-doses of MBP are thus considered to inhibit the transabdominal descent of the testis, probably due to its estrogenic activity, whereas low-doses of MBP may act as an anti-androgen and thereby inhibit inguinoscrotal testicular descent in postnatal rats.

Role of the gubernacular bulb in cremaster muscle development of the rat

The role of the gubernaculum during the inguino-scrotal phase of testicular descent remains controversial. Some authors propose involution and eversion while others suggest active migration, although the site of growth is unknown. We aimed to determine whether the gubernacular bulb is actively proliferating or regressing during inguino-scrotal testicular descent in the rat. Gubernacula were removed from Sprague-Dawley rats and congenitally-cryptorchid TS mutant rats. Animals (0, 3, 7, 10, and 11 days of age) were treated with bromodeoxyuridine (BUdR) 2 hr before they were killed. BUdR incorporation into newly synthesized DNA served as a marker of cell division. The gubernacula were histologically processed for hematoxylin-eosin (H&E) and immunoperoxidase staining. Four different areas within the gubernaculum were examined for BUdR-positive cells: area 1: plica gubernaculi (cord); area 2: pars infravaginalis gubernaculi (bulb); area 3: distal part of the cremaster muscle; and area 4: proximal part of the cremaster muscle. The rate of cell division for each of these areas was determined by counting the number of BUdR-positive cells per 100 cells. The highest rate of BUdR labeling in both types of rats was in area 2, which is the tip of the gubernacular bulb, and this was significantly greater (P < 0.0001) than in the gubernacular cord or developing cremaster muscle. The mitotic activity was also noted to be significantly greater (P < 0.0001) at the distal end of the cremaster muscle than at the proximal end. The amount of mitosis decreased significantly (P < 0.01) in areas 2 and 4 of the gubernaculum in Sprague-Dawley rats across the period studied. This trend was not observed in TS rats. Our results suggest that the bulb actively proliferates after birth, with possible differentiation into new cremaster muscle cells. We propose that the bulb is the growing end of the elongating gubernaculum, analogous to the growth of a limb bud. Anat Rec 267:159–165, 2002. © 2002 Wiley-Liss, Inc.

Role of the gubernacular bulb in cremaster muscle development of the rat

The role of the gubernaculum during the inguino-scrotal phase of testicular descent remains controversial. Some authors propose involution and eversion while others suggest active migration, although the site of growth is unknown. We aimed to determine whether the gubernacular bulb is actively proliferating or regressing during inguino-scrotal testicular descent in the rat. Gubernacula were removed from Sprague-Dawley rats and congenitally-cryptorchid TS mutant rats. Animals (0, 3, 7, 10, and 11 days of age) were treated with bromodeoxyuridine (BUdR) 2 hr before they were killed. BUdR incorporation into newly synthesized DNA served as a marker of cell division. The gubernacula were histologically processed for hematoxylin-eosin (H&E) and immunoperoxidase staining. Four different areas within the gubernaculum were examined for BUdR-positive cells: area 1: plica gubernaculi (cord); area 2: pars infravaginalis gubernaculi (bulb); area 3: distal part of the cremaster muscle; and area 4: proximal part of the cremaster muscle. The rate of cell division for each of these areas was determined by counting the number of BUdR-positive cells per 100 cells. The highest rate of BUdR labeling in both types of rats was in area 2, which is the tip of the gubernacular bulb, and this was significantly greater (P < 0.0001) than in the gubernacular cord or developing cremaster muscle. The mitotic activity was also noted to be significantly greater (P < 0.0001) at the distal end of the cremaster muscle than at the proximal end. The amount of mitosis decreased significantly (P < 0.01) in areas 2 and 4 of the gubernaculum in Sprague-Dawley rats across the period studied. This trend was not observed in TS rats. Our results suggest that the bulb actively proliferates after birth, with possible differentiation into new cremaster muscle cells. We propose that the bulb is the growing end of the elongating gubernaculum, analogous to the growth of a limb bud.

Enlargement of the processus vaginalis during testicular descent in rats

The role of the processus vaginalis (PV) during inguinoscrotal testicular descent remains controversial. Some authors propose passive dragging of the PV by the migrating testis, while others suggest active elongation. In addition, the exact site of growth is unknown. Our aim was to determine whether the PV actively proliferates at its tip or stretches passively during the inguinoscrotal phase of descent in the rat. Gubernacula were removed from Sprague-Dawley (SD) rats and congenitally-cryptorchid TS mutants. Animals (at days 3, 7, 10, and 11) were treated with bromo-uridine deoxyribose (BUdR) 2 h before death. BUdR incorporation into newly-synthesised DNA served as a marker for cell division. The gubernacula were processed for haematoxylin and eosin and immunoperoxidase staining. Three sites were examined: (1) the tip of the PV on either side of the gubernacular bulb; (2) the proximal gubernacular cord; and (3) the proximal parietal PV. At each site, 50 adjacent cells were counted and the number of positive cells recorded. The highest BUdR labelling in SD rats was at the tip (site 1) on day 3 (17/50) compared with sites 2 (11/50) and 3 (9/50) (P < 0.05). Labelling decreased by 7 and 11 days to similar levels in all three sites. In TS rats, labelling rates were lower at day 3 and were highest at the tip at day 11. These results suggest active growth of the caudal tip of the PV during testicular descent. In normal rats, the growth rate slows as the testis approaches the scrotum. By contrast, in TS rats growth continued longer. We propose that the PV elongates actively from the tip to allow the intraperitoneal testis to leave the abdomen in a special peritoneal diverticulum.

CONGENITAL UNDESCENDED TESTES IN NEONATAL PIGS AND THE EFFECT OF EXOGENOUS CALCITONIN GENE-RELATED PEPTIDE

Purpose We investigated the neonatal piglet as a possible animal model for cryptorchidism and to determine whether calcitonin gene-related peptide (CGRP), which has been proposed to regulate inguinoscrotal testicular descent, could induce testicular descent in piglets with congenital cryptorchidism. Materials and Methods We examined 38 cryptorchid piglets to document the anatomy in 8 and to investigate the role of CGRP in 30. The 2-week-old piglets were allocated randomly to receive a mini-osmotic pump containing CGRP at various concentrations or phosphate buffered saline. The pump was inserted surgically into the ipsilateral scrotum, with the contents blinded to the surgeon. The positions of the testes, pump and anatomical landmarks were measured and photographed. The pigs were sacrificed and dissected 2 weeks later, and the positions were remeasured and photographed. The testes were examined histologically. Results The 3 variants of cryptorchidism observed were intra-abdominal in 20 cases, inguinal in 9 and lateral inguinal ectopic in 9. CGRP had no effect on intra-abdominal or ectopic testes. In contrast, for inguinal testes exogenous CGRP caused a slight but significant 10 +/− 7.9 mm. descent towards the pump in 5 cases compared to −2.9 +/− 5.8 mm. in 4 controls. Conclusions Exogenous CGRP stimulated migration of inguinal testes that had been arrested in the line of descent while ectopic testes did not respond. These results support a role for CGRP in testicular descent and suggest that a slow release depot preparation might be useful as a possible treatment in some forms of cryptorchidism.

Does the gubernaculum migrate during inguinoscrotal testicular descent in the rat?

The role of the gubernaculum in descent of the testis is controversial. The mechanism of testicular descent has been studied in the rat, because inguino-scrotal descent occurs postnatally in this species. Several authors have claimed that the cremasteric sac forms by eversion of the gubernacular cone, whereby regression of the extra-abdominal part of the gubernaculum creates a space into which the gubernacular cone everts to form the processus vaginalis within the scrotum. This postulated lack of any gubernacular migration phase contrasts with the situation in the human, where gubernacular migration appears to be an integral component of testicular descent. This study was designed to determine in the rat whether there is any gubernacular migration toward the scrotum during testicular descent, or whether eversion of the cremasteric sac alone could account for the extension of this sac into the bottom of the scrotum. Oblique sagittal sections of the inguino-perineal region were taken from rats aged 21 days of gestation and days 1, 3, 4, 6, 8, and 10 days postnatally. Histological sections were examined and the following measurements were obtained: gubernacular cone height, gubernaculum-scrotum distance, processus vaginalis length, and pubic symphysis-anus distance. The gubernaculum was not in close proximity to the developing scrotum at any age. After 21 days of gestation, there was little evidence of a substantial gubernacular bulb distal to the processus vaginalis. At all ages the gubernacular cone height was significantly less than the distance from the gubernaculum to the scrotum. These results show that the gubernaculum does not develop in close proximity to the developing scrotum. Even if complete eversion of the gubernaculum takes place, the gubernaculum would still fail to reach the bottom of the scrotum. It is proposed that gubernacular eversion is more apparent than real and that some degree of gubernacular migration is needed for complete extension of the cremasteric sac to the bottom of the scrotum.