Wnt signalling in testicular descent: a candidate mechanism for cryptorchidism in Robinow syndrome.

BACKGROUND/AIMS: Robinow syndrome is caused by mutations in Wnt-5a or its receptor Ror2 and can lead to cryptorchidism, though the mechanisms are unclear. Wnt-5a knock-out mice fail to undergo gubernacular swelling, similar to insulin-like hormone 3 (INSl-3) knock-out mice. We aimed to characterise Wnt-5a and Ror2 expression in rat gubernacula to better understand how Wnt-5a signalling affects testicular descent. METHODS: Sprague-Dawley rats (n = 27) were collected with ethics approval (A644) at embryonic days (E) 15, 17, 19 and postnatal day (D) 2. Control and antiandrogen-treated groups were processed for immunohistochemistry for Wnt-5a, Ror2 and ß-catenin. Sagittal sections were examined using confocal microscopy. RESULTS: Wnt-5a and Ror2 were strongly expressed in the gubernacular bulb at E17 controls, their levels declining at E19 and almost absent by D2. Wnt-5a significantly co-localised with the important transcription factor ß-catenin at E17. There was no obvious difference in staining with androgen blockade. CONCLUSION: Wnt-5a, through Ror2 and ß-catenin may play a vital role in regulating the gubernacular swelling reaction downstream of INSL-3. Human mutations in Wnt-5a or Ror2 could prevent early gubernacular growth, as suggested by undescended testes in 70% of patients with Robinow Syndrome.

Regression of the mammary branch of the genitofemoral nerve may be necessary for testicular descent in rats.

PURPOSE: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Gubernaculum as icebreaker: do matrix metalloproteinases in rodent gubernaculum and inguinal fat pad permit testicular descent?

PURPOSE: Cryptorchidism is the most common male congenital abnormality. The rodent gubernaculum steers the testis from abdomen to scrotum postnatally by eversion and migration through the developing inguinal fat pad (IFP). We hypothesize that extracellular matrix remodeling in/around the gubernaculum is necessary for eversion and migration and is permitted by timed IFP maturation and aimed to examine regional development and matrix metalloproteinase (MMP) content. METHODS: Embryonic day 19 (E19) and postnatal days 0 and 2 (P0, P2) wild-type Sprague-Dawley rats (n = 10) were prepared for histologic examination (trichrome) and immunohistochemistry (membrane-type MMP-1 [MT1-MMP], MMP2) and analyzed using light/confocal microscopy. RESULTS: At E19, IFP contained fibroblasts and immature cells in an extensive collagenous extracellular matrix. Cells in the gubernaculum base were cytoplasmic-MT1-MMP-positive (inactive). At P0, the gubernaculum had everted, and adjacent cells were membranous-MT1-MMP-positive (active). At P2, the gubernaculum was migrating through the IFP, and adjacent cells were membranous-MT1-MMP-positive. Adipocyte maturation began cranially in the IFP and proceeded in a craniocaudal gradient until more uniformly mature at P2. CONCLUSION: The MT1-MMP-positive cells may remodel the gubernaculum for eversion and provide the collagenolysis necessary for migration, like an icebreaking ship, through the IFP, which matures to permit migration through collagen-rich tissue. Disruption of these processes may cause cryptorchidism.

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, ERα, and ERß 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 ERß. 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 ERß 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 ERß 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.

Gubernacular development in the mouse is similar to the rat and suggests that the processus vaginalis is derived from the urogenital ridge and is different from the parietal peritoneum.

BACKGROUND: Gubernacular development and testicular descent have been studied most extensively in rat models, but new transgenic mouse models require a deep understanding of normal mouse development so that results can be extrapolated to the human. We aimed to compare gubernacular anatomy during development in the mouse with that of the rat. METHODS: Time-mated mice (C57BL/6) and Sprague-Dawley rats were used to collect male fetuses at embryonic (E) days E13, E14, E15, E17, E18, and E19 and neonates at postnatal (P) days P0 and P2. Fetuses and newborn were processed for serial sections (sagittal, transverse, and coronal) and stained with hematoxylin and eosin, muscle markers (embryonic myosin, desmin), a neuronal marker (Tuj1), a mitotic marker (Ki67), and keratin marker to label epithelium. RESULTS: Early development of cremaster in the mouse was related to transversus abdominis muscle, but not internal oblique muscle (as in rats), and forms a monolaminar cremaster layer. There is close association between the regressing inguinal mammary bud and the gubernaculum in the mouse at E13. The peritoneal surface of the processus vaginalis (PV) covering the gubernaculum and epididymis was morphologically distinct from the remaining parietal peritoneum throughout development. CONCLUSIONS: Gubernacular development in mouse is similar to that in the rat except for certain structures, such as cremaster muscle. The PV seems to be derived from the surface of the urogenital ridge, separate from the remaining parietal peritoneum. This study suggests that the PV has evolved to aid testicular descent in this species, rather than a nondescript diverticulum of parietal peritoneum.

The development and anatomy of the gubernaculum in Hoxa11 knockout mice.

BACKGROUND: The gubernaculum is central to testicular descent, with recent evidence suggesting that it elongates to the scrotum like a limb bud. Homeobox (Hox) genes involved in limb bud outgrowth are expressed within the gubernaculum. Mice with homozygous Hoxa11 gene deletions have bilateral cryptorchidism. This study investigated the precise anatomical effects of Hoxa11 mutation on the mouse gubernaculum. METHODS: The pelvises of postnatal mice (n = 46; days 1-10) with Hoxa11 knockout (n = 19), heterozygotes (n = 11), and wild-type (n = 16) mice were serially sectioned and stained with hematoxylin and eosin. Immunohistochemistry was performed for the presence of desmin. RESULTS: Hoxa11 mutant mice had intraabdominal cryptorchid testes and highly convoluted vas deferentia. The gubernacular bulbs were abnormal, with no "outgrowth" and persistence of the prenatal "swelling reaction." Desmin immunostaining revealed the lack of undifferentiated mesenchymal cells usually seen as a "swirl" within the bulb and decreased formation of cremaster muscle. CONCLUSIONS: Hoxa11 may be involved in forming the growth center seen as the "swirl" of mesenchyme within the gubernacular bulb, consistent with these cells being required for gubernacular elongation during testicular descent. Hoxa11 mutations may well contribute to failure of gubernacular migration in boys with cryptorchidism.

Gender differences in reduced substance P (SP) in children with slow-transit constipation.

PURPOSE: Adult slow-transit constipation (STC) occurs predominantly in females and is associated with low numbers of substance P (SP)-containing nerves in colonic circular muscle. AIM: To determine if reduced SP nerves is female predominant in paediatric STC. METHODS: Children with STC were identified from records of more than 600 nuclear transit studies (NTS) and intestinal biopsies done for intractable chronic constipation between November 1998 and March 2009. Colonic seromuscular biopsies collected from hepatic and splenic flexures, and sigmoid colon were processed for immunohistochemistry. Nerve fibre density in circular muscle containing SP was measured qualitatively by a pathologist. RESULTS: Eighty-eight children with chronic constipation had both NTS and intestinal biopsies. Seventy-eight children (52 M; age 2-15.5 years; mean 7.7 years) had STC diagnosed by NTS. SP was reduced in 10/26 girls, but only 11/52 boys. CONCLUSION: In this sample, STC was more common in boys than girls. However, in girls with STC, SP deficiency occurred in 40%, when compared with 20% of boys. During puberty, the percentage of girls with reduced SP decreased, whilst the percentage of boys increased. These results suggest that STC is heterogeneous and that there are some gender differences, the implication of which requires further investigation.

Growth of the rat gubernaculum in vitro and localisation of its growth centre.

PURPOSE: Recent studies suggest that testicular descent is accomplished by outgrowth of the gubernaculum from the abdominal wall. The tip of the gubernaculum has been proposed as the primary site of growth, similar to an embryonic limb bud. We aimed to determine the maximum site of growth in organ culture. METHODS: Gubernacula from 1-day-old Sprague-Dawley rats (n = 40) were collected and divided into 4 groups as follows: whole gubernaculum (control), truncated gubernaculum (tip excised), gubernacular tip alone, and grafted gubernaculum with an extra tip on its side. Tissues were cultured with or without calcitonin gene-related peptide (CGRP) (714nmol/L) in medium for 24 hours. The area of each gubernaculum was determined by "Image J" analysis of digital photos collected via a Leica Wild M28 microscope (Leica Microsystems, Wetzler GmbH Germany) taken before and after culture. RESULTS: In organ culture, the neonatal rat gubernaculum normally shrank 10% to 15%, but this was prevented by the presence of exogenous CGRP (0.8% vs 11.8%; P < .003). By contrast, gubernacula with their tips excised were not affected by CGRP (3.4% vs 4.7%; not significant). Gubernacular tips alone did respond to CGRP (2.7% vs 13.5%; P < .03). Transplantation of the tip to another gubernaculum caused it to develop 2 tips. CONCLUSIONS: These results suggest that the rat gubernaculum contains a growth centre in its distal tip that can respond to CGRP. This is consistent with a limb bud model of gubernacular growth during the inguinoscrotal descent of the testis.

Evidence of a novel aggrecan-degrading activity in cartilage: Studies of mice deficient in both ADAMTS-4 and ADAMTS-5.

OBJECTIVE: To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Delta-cat) activity. METHODS: Femoral head cartilage from the joints of TS-4/TS-5 Delta-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1alpha (IL-1alpha) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE 373-374 ALG, SELE 1279-1280 GRG, FREEE 1467-1468 GLG, and AQE 1572-1573 AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo. RESULTS: TS-4/TS-5 Delta-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Delta-cat cartilage explants did not release aggrecan in response to IL-1alpha, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Delta-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid-stimulated cleavage at either EGE 373-374 ALG or AQE 1572-1573 AGEG. There was a low level of cleavage at SELE 1279-1280 GRG and major cleavage at FREEE 1467-1468 GLG. Ex vivo, cleavage at FREEE 1467-1468 GLG was substantially reduced, but still present, in TS-4/TS-5 Delta-cat mouse cartilage compared with wild-type mouse cartilage. CONCLUSION: An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1alpha. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E-G bonds but not E-A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

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 microm) 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 microm) 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.

Blocking aggrecanase cleavage in the aggrecan interglobular domain abrogates cartilage erosion and promotes cartilage repair.

Aggrecan loss from cartilage in arthritis is mediated by aggrecanases. Aggrecanases cleave aggrecan preferentially in the chondroitin sulfate-2 (CS-2) domain and secondarily at the E(373) downward arrow(374)A bond in the interglobular domain (IGD). However, IGD cleavage may be more deleterious for cartilage biomechanics because it releases the entire CS-containing portion of aggrecan. Recent studies identifying aggrecanase-2 (ADAMTS-5) as the predominant aggrecanase in mouse cartilage have not distinguished aggrecanolysis in the IGD from aggrecanolysis in the CS-2 domain. We generated aggrecan knockin mice with a mutation that rendered only the IGD resistant to aggrecanases in order to assess the contribution of this specific cleavage to cartilage pathology. The knockin mice were viable and fertile. Aggrecanase cleavage in the aggrecan IGD was not detected in knockin mouse cartilage in situ nor following digestion with ADAMTS-5 or treatment of cartilage explant cultures with IL-1 alpha. Blocking cleavage in the IGD not only diminished aggrecan loss and cartilage erosion in surgically induced osteoarthritis and a model of inflammatory arthritis, but appeared to stimulate cartilage repair following acute inflammation. We conclude that blocking aggrecanolysis in the aggrecan IGD alone protects against cartilage erosion and may potentiate cartilage repair.

Does chemical sympathectomy alter the ontogeny of gubernacular migration in vivo?

BACKGROUND: Testicular descent occurs in several stages, but the exact mechanism remains obscure. Sympathetic nerves have been proposed to have a role by a possible action on developing cremaster muscle, following observations of sympathetic dysfunction in cremaster from boys with cryptorchidism. This study aimed to see if chemical sympathectomy affected testicular descent in rats. METHODS: Sprague-Dawley dams were injected with 6-hydroxydopamine (days 15-19; 75 microg/kg) or control vehicle alone, and male pups examined at 0 to 10, 20, 30, and 60 days of age. The length of the processus vaginalis was measured and sections taken for histology. RESULTS: No difference in processus vaginalis growth was found between experimental and control groups, both macro- and microscopically. Chemical sympathectomy was confirmed by loss of adrenergic fibres in the adrenal cortex of experimental, but not control, animals. CONCLUSIONS: These studies suggest that sympathetic innervation is not an important part of testicular descent in the rat but does not exclude a pathologic role in undescended testes or effects in humans only.

A new role for androgen in testicular descent: permitting gubernacular cell proliferation in response to the neuropeptide, calcitonin gene-related peptide.

BACKGROUND/PURPOSE: Cell proliferation at the gubernacular tip increases in response to exogenous calcitonin gene-related peptide (CGRP) during migration into the scrotum. Calcitonin gene-related peptide is contained in the masculinized sensory branches of the genitofemoral nerve. We tested the independent effects of chemical sensory nerve disruption and prenatal androgen blockade on the in vitro gubernacular proliferative response to CGRP. METHODS: Neonatal Sprague-Dawley rats were injected with capsaicin, a sensory nerve toxin, and gubernacula dissected 2 days later (D2). Sprague-Dawley dams were injected with flutamide, an androgen receptor antagonist, between days 15 and 19 of gestation. Flutamide pretreated males, and normal neonatal rats, were dissected at D0 and D2. Gubernacula were cultured for 24 hours +/- CGRP, pulse-labelled for the last 4 hours of culture with bromodeoxyuridine, a thymidine analogue marker for DNA replication, sectioned, and stained using immunohistochemistry. The percentage of positively staining cells in the gubernacular tip was calculated from three separate counts by a blinded observer and compared using analysis of variance. RESULTS: Normal D0 gubernacular tips showed a significant response of cell proliferation to exogenous CGRP (34% vs 9% in controls, P < .001), which resolved by day 2 (16% vs 12%, P > .05). Calcitonin gene-related peptide markedly increased cell proliferation in D2 capsaicin pretreated gubernacula compared with controls (25% vs 14%, P < .01) and normal D2 gubernacula cultured with CGRP (P < .01). D0 flutamide pretreated cultured with CGRP showed no increase in cell proliferation compared with controls (16% vs 11%), but a small response was seen by D2 (19% vs 9%, P < .05). There was no significant difference between proliferation rates in the control groups. CONCLUSIONS: Sensory innervation interruption sensitises the gubernaculum to exogenous CGRP, suggesting upregulation of CGRP receptors. In contrast, androgen blockade abolishes the increased rate of cell proliferation within the gubernacular tip. We conclude that androgens are necessary to "preprogramme" the proliferative response of the gubernaculum to CGRP.

ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro.

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more 'aggrecanases' from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.

Gubernacular cell division in different rodent models of cryptorchidism supports indirect androgenic action via the genitofemoral nerve.

PURPOSE: The role of the "gubernaculum" in testicular descent remains controversial. Androgens are proposed to act indirectly by the genitofemoral nerve (GFN) releasing calcitonin gene-related peptide. The authors studied the effects of sensory nerve ablation and androgen blockade on mitosis in the gubernacular tip to determine whether androgens act directly or indirectly. METHODS: Five rat models were examined for bromodeoxyuridine (BUdR)-labeling: (i) Sprague-Dawley (SD) rats (controls), (ii) prenatal flutamide-treated rats (75 mg/kg to dams on D16-19 gestation), (iii) neonatal capsaicin-treated rats (50 mg/kg, subcutaneous on day 0), (iv) congenitally cryptorchid transcrotal (TS) rats, and (v) capsaicin-treated TS rats (50 mg/kg, subcutaneous on day 0). Newborn rats were collected at days 0, 2, 4, 6, 8, and 10 (age, n = 5/model, n = 30) and were injected intraperitoneally with 1 mg/kg BUdR, 2 hours before killing. Histological sections of gubernaculum were examined immunohistochemically for BUdR labeling. RESULTS: In SD (control) rats, DNA synthesis in the gubernacular tip was high at birth, reached a peak at day 2, and then decreased progressively until day 10. A similar pattern was observed in TS rats. However, quantitatively, the levels were significantly higher. In flutamide-treated rats, DNA synthesis was suppressed until day 6, similar suppression was observed in capsaicin-treated SD, and TS rats until day 4. CONCLUSIONS: Flutamide, a competitive androgen receptor blocker, reduces gubernacular mitosis to basal levels until day 6, highlighting the importance of androgen receptor. Excess DNA accumulation in TS rats is consistent with the known excess of GFN fibers and calcitonin gene-related peptide in this mutant. Capsaicin-inhibited mitosis in both day 2 SD and TS rats suggests that the GFN mediates androgen action on early postnatal gubernacular DNA synthesis and growth.

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.