Three-dimensional structure of testis cords in mice and rats.

BACKGROUND: Testis cord elongation and coiling, which occur in the final stage of testis formation, have been attributed to Sertoli cell proliferation; however, the underlying mechanisms remain unclear. OBJECTIVE: The aim of the present study was to clarify the precise three-dimensional structure of testis cords in the final stage of testis formation in mice and rats. MATERIALS AND METHODS: We reconstructed whole testis cords in the final stage of testis formation in mice (on embryonic days 15.5 and 18.5) and rats (on embryonic days 16.5 and 19.5) using serial paraffin sections and high-performance three-dimensional reconstruction software. RESULTS: Detailed morphometric parameters were calculated for three-dimensionally reconstructed testis cords in six mouse and rat testes each. The mean numbers of testis cords in mice and rats were 12.7 and 27.8, respectively. The mean number of branching points per testis cord was 1.52 in mice, whereas it was only 0.30 in rats. In contrast, the mean ratio of the inner cords, that is, cords not in contact with the tunica albuginea, was 23.0% in rats, whereas it was only 6.5% in mice. In both species, the cords on the cranial side coiled more strongly than those on the caudal side, consistent with the greater expansion of the testis volume on the caudal side. All cords formed right-handed helices from the rete testis side. DISCUSSION AND CONCLUSIONS: The present results suggest that testis cords undergo anastomosis at a higher frequency in mice than in rats and that the coiling of testis cords proceeds from the cranial to caudal side of the testis in both species.

Testis Cord Maintenance in Mouse Embryos: Genes and Signaling.

Testis cords, embryonic precursors of the seminiferous tubules, are fundamental for testis structure and function. Delay or disruption of testis cord formation could result in gonadal dysgenesis. Although mechanisms regulating testis cord formation during sex determination have been well-studied, the genes and signaling pathways involving in testis cord maintenance after the cords have formed are not well characterized. It is now clear that the maintenance of cord structure is an active process. In this review, we summarize the recent findings regarding the regulation of testis cord integrity by a series of Sertoli cell transcription factors, including the WT1-SOX8/SOX9-beta-CATENIN-DHH network, GPR56, STIM1, and NR0B1 (also known as DAX1). In particularly, we emphasize the underappreciated role of peritubular myoid cells in testis cord maintenance and their cooperation with Sertoli cells. The regulation of the size, shape, and number of testis cords by Sertoli cell proliferation (e.g., SMAD4, GATA4, and TGF-beta signaling), Leydig cell products (e.g., ACTIVIN A), vascular development (a lesson learned from PDGF signaling), and available gonad space (as observed in Ift144 mutant mice) is also addressed. Further efforts and new genetic models are needed to unveil the gene networks and underlying mechanisms regulating testis cord integrity and morphology after sex determination.

Germ cells influence cord formation and Leydig cell gene expression during mouse testis development.

BACKGROUND: It is widely accepted that, during the development of testes in the mammalian embryo, male germ cells are influenced by signals from the surrounding somatic cells, but not vice versa, so that germ cells are dispensable for the formation of testes. RESULTS: We now demonstrate that development of the mouse fetal testis is compromised in the absence of germ cells. Using two- and three-dimensional imaging techniques, we reveal that W(e)/W(e) mutant testes devoid of germ cells have misshapen and poorly organized cords. We also found that mutant gonads have fewer Sertoli cells than normal and that the Leydig cells express key markers at higher than normal levels. CONCLUSIONS: These observations point to the existence of germ cell-derived signals that directly or indirectly affect the Sertoli and Leydig cell populations, and provide a new paradigm for the organogenesis of the mammalian testes.

Homeoproteins Six1 and Six4 regulate male sex determination and mouse gonadal development.

The Y-linked gene Sry regulates mammalian sex determination in bipotential embryonic gonads. Here, we report that the transcription factors Six1 and Six4 are required for male gonadal differentiation. Loss of Six1 and Six4 together, but neither alone, resulted in a male-to-female sex-reversal phenotype in XY mutant gonads accompanied by a failure in Sry activation. Decreased gonadal precursor cell formation at the onset of Sry expression and a gonadal size reduction in both sexes were also found in mutant embryos. Forced Sry transgene expression in XY mutant gonads rescued testicular development but not the initial disruption to precursor growth. Furthermore, we identified two downstream targets of Six1/Six4 in gonadal development, Fog2 (Zfpm2) and Nr5a1 (Ad4BP/Sf1). These two distinct Six1/Six4-regulated pathways are considered to be crucial for gonadal development. The regulation of Fog2 induces Sry expression in male sex determination, and the regulation of Nr5a1 in gonadal precursor formation determines gonadal size.

The Wilms tumor gene, Wt1, maintains testicular cord integrity by regulating the expression of Col4a1 and Col4a2.

Wt1 is specifically expressed in Sertoli cells in the developing testis. A previous study has demonstrated that Wt1 plays a critical role in maintaining the integrity of testicular cords. However, the underlying mechanism is unclear. In this study, we found that the laminin-positive basal lamina lining the testicular cords was fragmented and completely absent in some areas of Wt1(-/flox); Amh-Cre testes, indicating that the testicular cord disruption can be attributed to the breakdown of the basement membrane. To explore the molecular mechanism underlying this effect, we examined the expression of cell adhesion molecules (CAMs) and testicular cord basal lamina components by real-time RT-PCR, Western blotting, and immunostaining. Compared with control testes, the expression of CAMs (such as E-cadherin, N-cadherin, claudin11, occludin, beta-catenin, and ZO-1) was not obviously altered in Wt1(-/flox); Amh-Cre testes. However, the mRNA level of Col4a1 and Col4a2 was significantly decreased in Wt1-deficient testes. Immunostaining assays further confirmed that the collagen IV protein levels were dramatically reduced in Wt1(-/flox); Amh-Cre testes. Moreover, luciferase and point mutation analyses revealed that the Col4a1 and Col4a2 promoters were additively transactivated by WT1 and SOX9. Given this finding and previous results showing that SOX9 expression declines rapidly after Wt1 deletion, we conclude that the loss of Wt1 in Sertoli cells results in the downregulation of the important basal lamina component, which in turn causes the breakdown of the basal lamina and subsequent testicular cord disruption.

Histological development of human testicular cords from 70 to 90 days of gestation.

The development of the testicular cord structure was investigated in 4 human fetuses between 70 and 90 days of gestation, in which the testicular cords are differentiating into the seminiferous tubules. Histological examinations were performed using stains with haematoxylin-eosin (HE), Masson's trichrome (MT), periodic acid schiff (PAS), anti-proliferating cell nuclear antigen (PCNA) monoclonal antibodies, and TUNEL methods. It was found that the testicular cords structures were indefinitely observed in HE-stained sections of four fetuses. However, the basement membranes of the testicular cord were clearly stained with MT, showing the tubular structure. Furthermore, cells in the testicular cords were positive with PAS, but the interstitial tissues outside the testicular cords were negative. PCNA-positive cells were detected not only inside but also outside the testicular cords, however, TUNEL positive cells are not detected throughout all testicular tissues.

A critical time window of Sry action in gonadal sex determination in mice.

In mammals, the Y-linked sex-determining gene Sry cell-autonomously promotes Sertoli cell differentiation from bipotential supporting cell precursors through SRY-box containing gene 9 (Sox9), leading to testis formation. Without Sry action, the supporting cells differentiate into granulosa cells, resulting in ovarian development. However, how Sry acts spatiotemporally to switch supporting cells from the female to the male pathway is poorly understood. We created a novel transgenic mouse line bearing an inducible Sry transgene under the control of the Hsp70.3 promoter. Analysis of these mice demonstrated that the ability of Sry to induce testis development is limited to approximately 11.0-11.25 dpc, corresponding to a time window of only 6 hours after the normal onset of Sry expression in XY gonads. If Sry was activated after 11.3 dpc, Sox9 activation was not maintained, resulting in ovarian development. This time window is delimited by the ability to engage the high-FGF9/low-WNT4 signaling states required for Sertoli cell establishment and cord organization. Our results indicate the overarching importance of Sry action in the initial 6-hour phase for the female-to-male switching of FGF9/WNT4 signaling patterns.

Peritubular myoid cells are not the migrating population required for testis cord formation in the XY gonad.

Cell migration is one of the earliest events required for development of the testis. Migration occurs only in XY gonads downstream of Sry expression and is required for the subsequent epithelialization of testis cords. Using organ culture experiments and tissue recombination, we and others speculated that peritubular myoid (PTM) cells were among the migratory cells and were likely the cell type required for cord formation. However, because no unique marker was found for PTM cells, their positive identification during or after migration remained unclear. alpha-Smooth Muscle Actin (alphaSma; approved gene symbol Acta2), a classic marker of adult PTM cells,is expressed broadly in testis interstitial cells at E12.5, and becomes highly enriched in PTM cells by E15.5-16.5. We used a novel transgenic line expressingEYFP under the control of an alphaSma promoter to determine whether alphaSma-EYFP positive cellsmigrate into the gonad. Surprisingly, mesonephroi expressing alphaSma-EYFP do not contribute any EYFP positive cells to XY gonads when used as donors in recombination cultures. These results indicate that alphaSma-EYFP cells do not migrate into the gonad during the critical window of sex determination and cannot be the migrating cell type required for testis cord formation. Our results suggest that PTM cells, and most other interstitial lineages, with the exception of endothelial cells, are induced within the gonad. These experiments suggest that endothelial cells are the migrating cell type required for epithelialization of testis cords.

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.

[Role of embryogenesis of the left renal vein and spermatic veins in etiopathogenesis of idiopathic varicocele]. / Ruolo della embriogenesi della vena renale sinistra e delle vene spermatiche nella etiopatogenesi del varicocele idiopatico.

The aim of this work was to clearly illustrate that the haemodynamic pathogenesis of the varicocele can be a consequence of a retrograde flux along the internal spermatic vein. The reflux is due to the use of a different path of drainage along the internal spermatic vein, which uses some collateral routes of the vessel, and mainly the nephrogenital branch. The diminishing pressure in the final segment of the internal spermatic vein is the focal points of the pathogenesis of the disease. The ontogenetic processes that lead to the formation of the left renal vein help in explaining the almost general one-sidedness of the disorder, its frequency and the fact that it is mostly the young who are affected by it. In fact, the variability of the timing and of the extension of the involution of the primitive venous system and of the processes of evolution which lead to the morphological definition of the tracts of the genital flow down, lead to the various manifestations of the idiopathic varicocele. This theory which also takes into account the physiological mechanisms of the venous drainage to the testicle in an anti-gravitation sense is part of the patterns that, under the diaphragm, rule the entire venous return of the blood toward to the heart.

Sex difference in target seeking behavior of developing cremaster muscles and the resulting first visible sign of somatic sexual differentiation in marsupial mammals.

Cremaster muscles are present in both male and female developing and adult marsupial mammals. They are complex structures and composed of several distinct bundles of striated muscle fibers provided with: (1) a distinct and extensive innervation; (2) a distinct blood vascular supply; (3) a distinct tendineous origin on the anterosuperior iliac spine; and (4) distinct target structures. The muscles thus seem to be separate anatomical entities and not a part of one or more of the layers of the ventral abdominal wall musculature. Cremaster muscles in males are elongated, are larger than in females, and for the most part are a component of the funiculus spermaticus. They insert on the distal part of the tunica vaginalis. The distal parts of the muscles in females are flattened ("fan shaped") and insert over a broad area on the dorsal borders of the mammary glands. Muscles in males have no relation whatsoever to the male mammary glandular rudiments. Muscles in females are attached at the base of the uterine round ligament. The remarkable sex difference in target structures of marsupial cremaster muscles becomes noticeable during perinatal life when outgrowing muscles take a different path in males and females. The initial appearance of this sexually dimorphic trait precedes the sexual differentiation of the genital ducts and external genitalia. In fetal males, the cremaster muscles grow in the direction of the site where scrotal bulges initially appear in the subcutaneous layers and later on the inguinal skin surface. They also take the gubernacular core of the ventral abdominal wall and the attached peritoneal epithelium with them during this outgrowth process. Consequently, this results in the development of a slitlike evagination of the abdominal lumen as the primary step to development of the processus vaginalis, while the testis and adjacent mesonephros and its duct are still attached to the posterior abdominal wall. In fetal females, the outgrowing cremaster muscles pass along the gubernacular core and, subsequently, this structure develops further as the tip (attached to the tubo-uterine junction) of the intra-abdominally protruding and further developing uterine round ligament. The female cremaster muscles grow further into caudal direction to shape a dorsal border of the developing mammary glands. The early onset of this sexually dimorphic outgrowth of cremaster muscles indicates that the "classical hormones" of sexual differentiation (anti-Müllerian hormone [AMH] and steroidal androgens) are not involved in this process. It could thus depend on primary genetic control with male development associated with the male-limited activity of genes on the Y-chromosomes and female development as the default process. Alternatively, the process in males could be under the control of an as yet unidentified third fetal testicular hormone involved in sexual differentiation processes which must then show an unexpectely early (i.e., perinatal) onset of its secretion.

Hydrocele of the spermatic cord: embryology and ultrasonographic appearance.

Five cases of hydrocele of the spermatic cord are presented. All five patients had a firm inguinal mass, and ultrasonography, performed to exclude adenopathy, incarcerated hernia, and paratesticular tumor, showed an avascular cystic mass superior to and separate from the testicle. A testicular hydrocele did not coexist. This typical appearance should lead to a confident diagnosis of this benign but rare anomaly. Elective surgery may prevent the development of an acquired indirect hernia.

Retinoic acid affects basement membrane formation of the seminiferous cords in 14-day male rat gonads in vitro.

The vitamin A derivative retinoic acid (RA) has been previously shown to have teratogenic effects and an ability to modulate cell differentiation in vivo and in vitro. In this study bilateral testicular primordia with the mesonephroi attached were isolated from rat fetuses at 14.5 days of gestation. The gonads were cultured on agar-coated grids in a synthetic medium. RA was added to male rat embryonic gonad cultures at a final concentration of 10(-6) M for 3 h. Two types of controls were prepared: (1) by omitting RA from the culture medium (alcohol controls) and (2) by using plain medium (untreated controls). When applied to gonad cultures RA was found to affect basement membrane development and disturb the general appearance of the tissue. All controls exhibited normal morphology. In order to evaluate the morphological changes observed due to the RA treatment, constituents of the basement membrane, laminin and collagen IV, were localized immunohistochemically at the light microscope level. Basement membrane was also studied at the electron microscope level in control and RA-treated cultures. We propose that one of the effects RA has on rat testicular morphogenesis is the irreversible suppression of seminiferous cord basement membrane formation and the disruption of normal testicular morphogenesis.

[Adrenal ectopia in the spermatic cord]. / Ectopia adrenal en cordón espermático.

As a result of finding some ectopic adrenal debris in the spermatic cord, the present report studies the embryogenesis and clinical implications that can derive from the existence of the referred adrenal debris. The relevant literature is reviewed and six new cases contributed, one of them bilateral.

Fine structural characteristics of testicular cord formation in the developing rabbit gonad.

This paper presents morphological (light- and electron-microscopical) evidence for the role of the mesonephros in contributing cells to the differentiating indifferent gonad and, after sexual differentiation, to the testis. A continuous process is revealed during which segregation of cells occurs from the developing and regressing mesonephros. Additionally, the complementary role of the coelomic epithelium in gonadal ridge and testis formation is demonstrated. The differentiation of testicular cords, their remodelling from a primary reticulum, and the composition and further change of the cellular content during the period after sexual differentiation is described using a computer-aided three-dimensional reconstruction system. Apart from these morphogenetic events, cytodifferentiation in the somatic cells of the indifferent gonad and of the early differentiated testis is demonstrated using indirect immunofluorescence in combination with monoclonal antibodies to the intermediate filament proteins keratin 8 and 18 and vimentin. The immunohistochemical results show that different forms of cytodifferentiation coexist among the somatic cells present in the indifferent gonad and in the testis early after sexual differentiation.

Morphogenesis of the gubernaculum testis in Sprague-Dawley rat. Preliminary report.

The morphology of testicular descent in the Sprague-Dawley rat from day 14 of gestation to newborn was studied; the purpose was to analyse the anatomy and related observations made with light and scanning microscopy. Adult female rats were mated during estrus. The presence of spermatozoa in vaginal smears was taken to indicate day 0 of gestation. Animals were kept in a controlled 12-hour light-darkness cycle; food and water were provided ad libitum. At day 14 genital ducts are similar in both sexes. At day 15 and 16 the gubernaculum testis is seen as a mesenchymal cord that extends from mesonephros to the pelvic floor. At day 17 near the enlarged caudal end of gubernaculum, evagination of the abdominal cavity begins forming the peritoneo-vaginal process. The testis ligament as well as the gubernaculum testis attaching to the epididymis at different locations have no direct relation between themselves as such. During day 18, testis rotates on its own longitudinal axis in a ventral-lateral direction. This movement twists the testis ligament, the epididymis and deferent duct lengthen and these structures are lined up in the direction of the peritoneo-vaginal process. The testis is generally situated in the newborn, on or near the internal ring. The gubernaculum testis is attached to the epididymis but does not exercise traction on the testis. The epididymis is a factor in this descending process.

Tubular embryonal remnants in the human spermatic cord.

The spermatic cords (SCs) obtained from: (a) autopsies of 67 adults and 50 children; (b) 3 fetuses; (c) 12 surgical specimens from 9 adults and 3 children with testicular tumors, and (d) 26 surgical specimens (14 adults and 12 children) with diagnosis of SC cysts, were studied by light microscopy. The histological study revealed the occurrence of tubular embryonal remnants (TERs) in 19 autopsied adults, 14 autopsied children, 3 fetuses, 2 surgical specimens (1 adult and 1 child) owing to testicular tumors, and 6 surgical specimens with diagnosis of SC cysts. The TERs were found in the low, middle or high thirds of the SC and consisted of a cuboidal or columnar, often ciliated epithelium surrounded by connective tissue. In the 3 fetuses and in 3 autopsied children renal glomeruli were found in close relation with the TERs. In 2 cases of SCs showing cysts and TERs both structures contained spermatozoa. The histological pattern of the TERs suggest that they are wolffian derivatives which might give rise to SC cysts.

Histological classification of spermatic cord cysts in relation to their histogenesis.

Light microscopy study of spermatic cord cysts in 26 men revealed three different histological patterns. First, cysts of probable mesothelial origin (14 cases) with an unilocular aspect: their epithelial cells showed poor cohesion and often appeared sloughed; subepithelial hyalinization or fibrin deposits were frequent. Second, cysts of probable embryonal (mesonephric) origin (8 cases): they were usually multilocular cysts and their epithelial cells showed great cohesion: zones of ciliated columnar epithelium associated with embryonal remnants displaying a similar epithelium were often found; the embryonal remnants and the cyst lumen contained spermatozoa in 2 cases. Third, cysts of doubtful origin (4 cases) showing abundant inflammatory infiltrates, which had destroyed the epithelium; the unilocular pattern observed in 3 cases suggests a mesothelial origin for these cysts, while the multilocular pattern and presence of embryonal remnants in the other case suggest an embryonal origin.