Loss of WNT4 in the gubernaculum causes unilateral cryptorchidism and fertility defects.

Undescended testis (UDT) affects 6% of male births. Despite surgical correction, some men with unilateral UDT may experience infertility with the contralateral descended testis (CDT) showing no A-dark spermatogonia. To improve our understanding of the etiology of infertility in UDT, we generated a novel murine model of left unilateral UDT. Gubernaculum-specific Wnt4 knockout (KO) mice (Wnt4-cKO) were generated using retinoic acid receptor ß2-cre mice and were found to have a smaller left-unilateral UDT. Wnt4-cKO mice with abdominal UDT had an increase in serum follicle-stimulating hormone and luteinizing hormone and an absence of germ cells in the undescended testicle. Wnt4-cKO mice with inguinal UDT had normal hormonal profiles, and 50% of these mice had no sperm in the left epididymis. Wnt4-cKO mice had fertility defects and produced 52% fewer litters and 78% fewer pups than control mice. Wnt4-cKO testes demonstrated increased expression of estrogen receptor α and SOX9, upregulation of female gonadal genes, and a decrease in male gonadal genes in both CDT and UDT. Several WNT4 variants were identified in boys with UDT. The presence of UDT and fertility defects in Wnt4-cKO mice highlights the crucial role of WNT4 in testicular development.

Role of Gubernaculum testis inervation during the process of testicular migration in human fetuses.

PURPOSE: The gubernaculum seems to be the most important anatomical structure in the testicular migration process. The objective of this paper is to review current literature regarding the role of gubernaculum testis nerves in testicular migration. We conducted a comprehensive literature review about the gubernaculum testis innervation. A PubMed database search was performed in April 2024, focusing on gubernaculum testis and cryptorchidism and genitofemoral nerve (GFN) and calcitonin gene-related peptide (CGRP) gene. The gubernaculum has its own nerve supply, the GFN, descending on the anteromedial surface of the psoas muscle from L1-L2 segments. The second phase of testicular descent is regulated by androgens and CGRP, released from the sensory nucleus of the GFN. The GFN doesn't directly play a role in testicular migration but there is a theory that shows a regulatory function of this nerve in hormonal action during this process. The gubernaculum testis has important structural alterations during the testicular migration and the genitofemoral nerve and CGRP gene are of great importance in this process. The genitofemoral nerve provides motor innervation to the cremaster muscle and gubernaculum, which helps regulate the position of the testes within the scrotum.

The RXFP2-PLC/PKC signaling pathway mediates INSL3-induced regulation of the proliferation, migration and apoptosis of mouse gubernacular cells.

BACKGROUND: Testicular hypoplasia can affect the sexual and reproductive ability in adulthood, and even increase the risk of cancer. Abnormal development of the gubernaculum is one of the important factors of testicular hypoplasia. Therefore, a study of the structure and function of the gubernaculum is an important but neglected new breakthrough point for investigating the normal/abnormal development of the testis. Previous findings showed that Insulin like factor 3 (INSL3) is a key factor regulating the growth of gubernaculum, however, the mechanism by which INSL3 acts on the gubernaculum remains unknown. Therefore, we probed the mechanism associated with INSL3-induced the proliferation, migration, and apoptosis of gubernacular cells in mice. METHODS: A culture cell model of neonatal mice gubernaculum is established by INSL3 intervention. We blocked PLC/PKC signaling pathway with U73122 pretreat to investigate the role of the PLC/PKC signaling pathway. The changes of cell proliferation, migration, and apoptosis were detected by molecular biological methods. In addition, the levels of PCNA and F-action were detected by immunofluorescence and western blotting. RESULTS: We found that INSL3 can promote the proliferation and migration of gubernacular cells and inhibit their apoptosis, meanwhile, INSL3 significantly up-regulated PLC/PKC protein phosphorylation. However, treatment with the PLC/PKC signaling pathway inhibitor U73122 significantly inhibited these effects of INSL3. Besides, we found that INSL3 could up-regulate the protein expression level of PCNA and F-actin, while the PCNA and F-actin expression was significantly weakened after U73122 pretreatment. CONCLUSIONS: This research revealed that INSL3 binding to RXFP2 may up-regulate the expression levels of PCNA and F-actin by activating the PLC/PKC signaling pathway to promote the proliferation and migration of gubernacular cells. It suggests that the RXFP2-PLC/PKC axis may serve as a novel molecular mechanism by which INSL3 regulates growth of the gubernaculum.

Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals.

Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These "molecular vestiges" provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

Gubernaculum Testis and Cremasteric Vessel Preservation during Laparoscopic Orchiopexy for Intra-Abdominal Testes: Effect on Testicular Atrophy Rates.

PURPOSE: Gubernaculum sparing laparoscopic orchiopexy, which involves anatomical delivery of the testis through the internal inguinal ring, has been proposed as an alternative to conventional laparoscopic Fowler-Stephens orchiopexy, maximizing collateral blood supply and potentially decreasing atrophy rates. We compared the 2 techniques to test this hypothesis. MATERIALS AND METHODS: The primary (dependent) outcome of the study was rate of testicular atrophy, which was defined as palpation of a nubbin or inability to palpate a testis (complete atrophy) on postoperative physical examination at 3 and 12 months. Doppler ultrasound was obtained routinely to further confirm the diagnosis of testicular atrophy. Independent variables that were captured were age at surgery, type of procedure (conventional laparoscopic Fowler-Stephens orchiopexy vs gubernaculum sparing laparoscopic orchiopexy), surgical approach (single vs 2-stage), location of intra-abdominal testis (high vs low) and patency of the internal inguinal ring. RESULTS: Mean ± SD age at surgery was 25.7 ± 13.3 months (median 22). Laparoscopy was carried out for nonpalpable testes and revealed vanishing intra-abdominal testes in 120 cases (29%), peeping testes in 80 (19%) and intra-abdominal testes in 212 (51%), with 104 being low and 108 being high in the abdomen. A single stage procedure was performed in 44 cases (21%) and a 2-stage procedure in 168 (79%). Based on surgeon preference, conventional laparoscopic Fowler-Stephens orchiopexy was undertaken in 46 patients (22%) and gubernaculum sparing laparoscopic orchiopexy in 166 (78%). Overall testicular atrophy rate was 6.6% (14 of 212 cases). Atrophy was observed in 13 of 46 testes after conventional laparoscopic Fowler-Stephens orchiopexy and 1 of 166 following gubernaculum sparing laparoscopic orchiopexy (28.3% vs 0.6%, p <0.01). CONCLUSIONS: Gubernaculum sparing laparoscopic orchiopexy is a feasible alternative to conventional laparoscopic Fowler-Stephens orchiopexy. Our findings suggest that preservation of additional vascular supply to the testis (cremasteric vessels and deferential artery) may translate into improved testicular survival rates following laparoscopic orchiopexy.

Diethylstilbestrol regulates mouse gubernaculum testis cell proliferation via PLC-Ca2+ -CREB pathway.

Recent evidence suggested a positive correlation between environmental estrogens (EEs) and high incidence of abnormalities in male urogenital system, but the mechanism remains unclear. Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen that disrupts the morphology and proliferation of gubernaculum testis cells, but the underlying mechanism is unclear. In this study, mouse gubernaculum testis cells were pretreated with phospholipase C (PLC) inhibitor U-73122 and then treated with DES. The results demonstrated that U-73122 impaired DES-evoked intracellular Ca2+ mobilization in gubernaculum testis cells and inhibited DES-induced proliferation of gubernaculum testis cells. Mechanistically, we found that U-73122 inhibited DES-induced activation of cAMP-response element binding protein (CREB) in gubernaculum testis cells. In conclusion, these data suggest that the effects of DES on mouse gubernaculum testis cells are mediated by PLC-Ca2+ -CREB pathway. SIGNIFICANCE OF THE STUDY: Environmental estrogens remain a serious threat to male reproductive health, and it is important to understand the mechanism by which EEs affect the male productive system. Here we explore potential mechanisms how the proliferation and contractility of gubernaculum testis cells are regulated by diethylstilbestrol. Our findings provide the first evidence that PLC-Ca2+ -CREB signalling pathway mediates the nongenomic effects of diethylstilbestrol on gubernaculum testis cells. These findings provide new insight into the role of diethylstilbestrol in the aetiology of male reproductive dysfunction and will help develop better approaches for the prevention and therapy of male reproductive malformation.

Anatomy and histology of the scrotal ligament in adults: inconsistency and variability of the gubernaculum testis.

PURPOSE: The anatomy of gubernaculum testis (GT) is often discussed; however, the postnatal anatomy of the GT or scrotal ligament (SL) is rarely described. Hence, we performed an anatomical and histological study to analyze histologically the structures between testis and scrotum. METHODS: We performed anatomical dissections on 25 human fresh cadavers' testes. Each testis was removed with its envelopes and macroscopically analyzed. Then samples were included for histological study. Finally, they were analyzed under microscope, looking for attachments between testis, epididymis and scrotal envelopes. RESULTS: The absence of proximal and distal attachment was found in 56.0% of cases. Looking at the proximal attachment of the SL, the main one found is the epididymal attachment (28.0%), whereas no cases of testis attachment was found. Distally, there are more variations with scrotal attachment (12%) and cremaster attachment (12.0%). We found a significant prevalence of multiple adherences in 16.0% of cases too. Finally, in 15 cases (57.7%) an attachment is present between testis and epididymis, as it is commonly described. CONCLUSIONS: In the majority of cases there is no attachment of the lower pole of the testis and epididymis and these structures remain free. So it seems that the SL disappears with aging. Moreover, there is not only one kind of ligamentous attachment, but a high variability of attachments at the lower pole of the testiculo-epididymal structure. When it exists, this structure is never a real ligament and it seems more appropriate to use the term "attachments".

New insights into the morphogenesis of the gubernaculum testis and the inguinal canal.

There is no consensus about the role of the gubernaculum testis (GT). Nineteen human embryos (Carnegie stages 15-23), 36 fetuses (9 weeks to term), and eight neonates were examined. All the embryos and 25 fetuses (from weeks 9-16) were processed for paraffin wax histology and serially sectioned at 10 µm thickness. The remaining 11 fetuses and the eight neonates were fresh specimens that were dissected under a surgical microscope. The GT appeared during the embryonic period (stages 17-23) with a horseshoe-shaped mesenchyme condensation of the superior concavity, which was observed in four different topographical regions sequentially through development. The GT was not attached at either of its ends in any of the specimens, not to the gonad or in the scrotal sac. The inguinal canal differentiates around the inguinal portion of the GT during the late embryonic period. After stage 21, the GT appears enveloped by an evagination of the peritoneal cavity. It has few striate muscular fibers and vessels. Although the GT does not appear to have the role traditionally attributed to it, it is still an essential structure and indirectly facilitates the descent of the testes. It contributes to the formation of the inguinal canal and then forges the pathway that the testes will subsequently take through the inguinal canal as they migrate from the abdominal cavity into the scrotal sac. Clin. Anat. 30:599-607, 2017. © 2017 Wiley Periodicals, Inc.

[Impacts of DES on the expressions of related genes in the gubernaculums testis of newborn mice].

OBJECTIVE: To investigate the influence of diethylstilbestrol (DES) on the mRNA expressions of the androgen receptor (AR), estrogen receptor α (ERα), proliferating cell nuclear antigen (PCNA), and actin alpha 1 (ACTα1) in the gubernaculums testis of newborn mice and explore their action mechanisms. METHODS: A total of 140 male Kunming mice were randomly divided into a blank control, a dimethyl sulfoxide (DMSO) control, and 5 experimental groups to be treated subcutaneously with normal saline, DMSO, and DES at 0.02, 0.1, 0.5, 10 and 50 µg per kg of the body weight per day, respectively, at gestation days 9－17. On the first day after birth, the animals were sacrificed and the gubernaculums testis collected for detection of the mRNA expressions of AR, ERα, PCNA and ACTα1 by RT-PCR. RESULTS: Compared with the DMSO control, the experimental groups, particularly the DES 10 and 50 µg groups, showed significant increases in the mRNA expression of ERα (RE2 = 0.825, P <0.05), but remarkable decreases in those of AR, PCNA and ACTα1 (RA2 = 0.713, RP2 = 0.946, RT2 = 0.960, P <0.01), all in a dose-dependent manner. CONCLUSIONS: The AR, ERα, PCNA, and ACTα1 mRNA are expressed in the gubernaculum testis of normal newborn mice, and their expression levels may be influenced by intervention with different concentrations of DES during the gestation. Exogenous estrogens may affect the proliferation and contraction of gubernaculum testis cells and consequently the normal development of the testis or even the whole male reproductive system by influencing the metabolism of ER and/or AR.

Fetal Rat Gubernaculum Mesenchymal Cells Adopt Myogenic and Myofibroblast-Like Phenotypes.

PURPOSE: Gubernaculum-cremaster complex development is hormonally regulated and abnormal in a cryptorchid rat model. Using cell tracking techniques and imaging we studied myogenic phenotypes and fates in the fetal rat gubernaculum-cremaster complex. MATERIALS AND METHODS: Embryonic day 17 gubernaculum-cremaster complexes were labeled with CellTracker™ or the DNA synthesis marker EdU (5-ethynyl-2'-deoxyuridine), or immobilized in Matrigel® and grown in culture. Embryonic day 17 to 21 gubernaculum-cremaster complex sections and cells were imaged using wide field and deconvolution immunofluorescence microscopy, and muscle and/or myofibroblast specific antibodies. Deconvolved image stacks were used to create a 3-dimensional model of embryonic day 21 gubernaculum-cremaster complex muscle. RESULTS: PAX7 (paired box 7) positive and myogenin positive muscle precursors were visible in a desmin-rich myogenic zone between muscle layers that elongated and became thicker during development. Gubernaculum-cremaster complex inner mesenchymal cells expressed desmin and αSMA (α smooth muscle actin) at lower levels than in the myogenic zone. After pulse labeling with CellTracker or EdU mesenchymal cells became incorporated into differentiated muscle. Conversely, mesenchymal cells migrated beyond Matrigel immobilized gubernaculum-cremaster complexes, expressed PAX7 and fused to form striated myotubes. Mesenchymal gubernaculum-cremaster complex cell lines proliferated more than 40 passages and showed contractile behavior but did not form striated muscle. Our 3-dimensional gubernaculum-cremaster complex model had 2 orthogonal ventral layers and an arcing inner layer of muscle. CONCLUSIONS: Our data suggest that mesenchymal cells in the peripheral myogenic zone of the fetal gubernaculum-cremaster complex contribute to formation of a distinctively patterned cremaster muscle. Nonmyogenic, desmin and αSMA positive gubernaculum-cremaster complex mesenchymal cells proliferate and have a myofibroblast-like phenotype in culture. Intrinsic mechanical properties of these divergent cell types may facilitate perinatal inversion of the gubernaculum-cremaster complex.

The adenomatoid odontogenic tumour: an update of selected issues.

The aim of this update was to present the recent notable progress within remaining questions relating to the adenomatoid odontogenic tumour (AOT). Selected issues that were studied included the following: (i) AOT history and terminology, (ii) the so-called peripheral AOT, (iii) AOT and the gubernaculum dentis and (iv) the so-called adenomatoid odontogenic cyst (AOC). The earliest irrefutable European case of AOT was described in 1915 by Harbitz as 'cystic adamantoma'. Recently, Ide et al. have traced two Japanese cases with irrefutable proof described by Nakayama in 1903. The so-called peripheral (gingival) variant of AOT seems to cover a dual pathogenesis, both an 'erupted intraosseous' and an 'extraosseous' (gingival) one. In 1992, we theorized that the generally unnoticed gubernaculum dentis (cord and canal) seems to be involved in the development of AOT. Ide et al. have concluded that the dental lamina in the gubernacular cord seems to be an embryonic source of the vast majority of AOTs. The suggestion by Marx and Stern to change the nomenclature of AOT to adenomatoid odontogenic cyst (AOC) is critically discussed. The present authors agree on the background of the work of several groups of researchers and WHO/IARC classifications that the biology of the follicular variant of AOT is already fully explained and does not make room for any change in diagnostic terms. Further, there is no reason to change terminology in this case where improvements or conditions to better clinical management are not an issue.

Gubernaculum-sparing orchiopexy versus gubernaculum excision; A randomized trial on success and atrophy rates.

INTRODUCTION AND OBJECTIVE: Previous studies showed that extra blood supply can decrease testicular atrophy following laparoscopic orchiopexy. We evaluated the impact of preserving the gubernacular attachment (which contains blood supply from cremasteric artery and its anastomoses) on atrophy rates following open conventional orchiopexy. STUDY DESIGN: This double-blinded randomized trial was implemented from March 2022 to September 2023. Included boys with non-palpable testis, even with examination under anesthesia, underwent diagnostic laparoscopy to evaluate the testis's location and size. Nubbin testes and those with > 2-cm distance from the internal inguinal ring. Participants were assigned into two groups (gubernaculum sparing (GS) and excision (GE)) by permuted block randomization. Overall success was defined as achieving both morphologic success (atrophy <20% of the intraoperative size) and anatomical success (scrotal or high-scrotal locations). Boys were followed at three- and six-month post-surgery via ultrasound. Independent t-test, repeated ANOVA, and Friedman's tests were used where appropriate. RESULTS: Of 92 boys (105 UDTs overall), 75 testes (36 in GS, 39 in GE groups) were used in the analysis. The mean age of participants was 25 ± 17 months (range 6-84). The mean testis size of cases intraoperatively was 460 ± 226, 396 ± 166, and 520 ± 258 mm3 among all participants, GS, and GE cases, respectively. Both groups showed a significant decrease in testicular volume on both follow-up checkpoints, but this decrement was significantly higher in the GE group (p < 0.001). The anatomical success rate was significantly higher among GS boys (97.2% versus 82.1%; p = 0.038). The overall success rate was significantly higher for the GS group (61.1% versus 25.6%; p = 0.002). CONCLUSION: Although mean testicular volume decreased in both groups, we found superior morphologic and overall success rates among the GS group. The greatest size reduction was noted at the three-month post-surgery compared to the six-month checkpoint. TRIAL REGISTRATION: https://irct.ir/trial/58842.

Review of Euchromadorinae (Nematoda: Chromadorida) with description of a new species of Trochamus Boucher & De Bovée, 1971 from the Sundarban, India.

Diagnostic characters for all 11 valid genera of Euchromadorinae are presented with taxonomic key on the basis of morphology of male copulatory apparatus, cuticular pattern, amphideal fovea, and buccal onchia. The key to the species of Trochamus spp. is also constructed with the description of T. timmi sp. n. from the mud-flat of Sundarban, India. The newly described species is different from other Trochamus spp. on the basis of the appearance of lateral differentiation of cuticle, long curved spicule, simple gubernaculum and the presence of pre-cloacal modification in male.

Three new free-living marine nematode species of Subsphaerolaimus Lorenzen, 1978, Halichoanolaimus de Man, 1886 and Belbolla Andrssy, 1973 from mangrove wetlands in Taiwan.

Three new species of free-living marine nematodes belonging to the genera Subsphaerolaimus, Halichoanolaimus and Belbolla are described from the mangrove wetlands of western Taiwan Island. Subsphaerolaimus danshuiensis sp. nov. is characterized by a body length of 13451693 m, subcephalic setae 22.565.0 m long, cervical setae 16.533.0 m long, an L-shaped spicule 66.976.4 m long, and a gubernaculum with a caudally-dorsally directed apophysis 16.423.0 m long. Halichoanolaimus sicaoensis sp. nov. is characterized by an amphidial fovea with 3.53.75 turns, a conico-cylindrical tail with the cylindrical portion approximately 3/4 of the total tail length, and 1314 not equidistant papillose precloacal supplements. Belbolla forkyspicula sp. nov. is characterized by seven oesophageal bulbs, a short tail, a spicule with a proximal fork, and two winged supplements. Differentiating characteristics of the genera Subsphaerolaimus, Halichoanolaimus and Belbolla are provided. Types are deposited in the College of Fisheries, Jimei University.

Imaging characteristics of the gubernaculum tracts in successional teeth related to deciduous fused teeth on computed tomography.

The purpose of this study was to elucidate the imaging characteristics of the gubernaculum tracts in successional teeth related to fused deciduous teeth on computed tomography. The imaging findings of 15 gubernaculum tracts in successional teeth related to fused deciduous teeth were retrospectively analyzed using cone-beam computed tomography or multidetector computed tomography. In cases without a congenitally defected successor, the two gubernaculum tracts of two successional teeth related to fused deciduous teeth were fused into one. Gubernaculum tracts (GTs) in mesial successors were vertical, but in distal successors they were inclined to mesial. The major abnormalities of the successional teeth related to fused deciduous teeth were delayed eruption and delayed formation. No inclined mesial successors were found, whereas most of the distal successors were inclined to mesial along with the inclined GT. The gubernaculum tracts of successors with a congenital defect of the other successors were vertical, and such successors had no abnormalities. The present study showed the imaging characteristics of gubernaculum tracts in successional teeth related to fused deciduous teeth. The abnormal eruption of successional teeth related to fused deciduous teeth may be associated with the characteristics of their gubernaculum tracts.

Open Versus Laparoscopic Gubernaculum-Sparing Second-Stage Fowler-Stephens Orchiopexy for Intra-Abdominal Testis: A Long-Term Study.

Background: The benefits of performing open versus laparoscopic gubernaculum-sparing second-stage Fowler-Stephens orchiopexy (FSO) remain unclear. We compared the two techniques to answer this question. Methods: We retrospectively studied a cohort of patients who underwent laparoscopic first-stage FSO and open versus laparoscopic gubernaculum-sparing second-stage FSO at our institution between September 2004 and June 2020 (all patients underwent surgery by a single surgeon). We evaluated both procedures based on the incidence of testicular atrophy, testicular ascent, and other complications. Results: The age at initial surgery was 45.7 ± 28.2 months (median, 39). One hundred nine cases were treated with open second-stage gubernaculum-sparing FSO (OFSO), and 96 cases were treated with laparoscopic second-stage gubernaculum-sparing FSO (LFSO). The mean follow-up period was 59.8 months (median, 54; standard deviation, +35). The overall testicular atrophy rate was 1.5%. Atrophy was observed in 2 and 1 patient in the OFSO and LFSO groups, respectively (1.8% versus 1.0%, P > .05). There was no significant difference in the incidence of testicular ascent between both groups (2.8% versus 3.1%). Five and four complications were noted in the OFSO and LFSO groups, respectively (P > .05). Conclusions: Second-stage gubernaculum-sparing FSO achieved high testicular survival rates and satisfactory testicular positions. Neither the open nor laparoscopic approach appeared superior, because the overall testicular survival rates and incidence of testicular ascent and other complications were equivalent between both groups.