Anatomy, Endocrine Regulation, and Embryonic Development of the Rete Testis.

Reproduction in males requires the transfer of spermatozoa from testis tubules via the rete system to the efferent ductules, epididymis, and vas deferens. The rete therefore forms an essential bridging system between the testis and excurrent ducts. Yet the embryonic origin and molecular regulation of rete testis development is poorly understood. This review examines the anatomy, endocrine control, and development of the mammalian rete testis, focusing on recent findings on its molecular regulation, identifying gaps in our knowledge, and identifying areas for future research. The rete testis develops in close association with Sertoli cells of the seminiferous cords, although unique molecular markers are sparce. Most recently, modern molecular approaches such as global RNA-seq have revealed the transcriptional signature of rete cell precursors, pointing to at least a partial common origin with Sertoli cells. In the mouse, genes involved in Sertoli cell development or maintenance, such as Sox9, Wt1, Sf1, and Dmrt1, are also expressed in cells of the rete system. Rete progenitor cells also express unique markers, such as Pax8, E-cadherin, and keratin 8. These must directly or indirectly regulate the physical joining of testis tubules to the efferent duct system and confer other physiological functions of the rete. The application of technologies such as single-cell RNA-seq will clarify the origin and developmental trajectory of this essential component of the male reproductive tract.

Morphology of mouse seminiferous tubules.

We developed a technique to analyze the high-resolution three-dimensional (3D) structure of seminiferous tubules. It consists of segmentation of tubules in serial paraffin sections of the testis by marking the basement membrane with periodic acid-Schiff or a fluorescent anti-laminin antibody followed by 3D reconstruction of tubules with high-performance software. Using this method, we analyzed testes from mice at different ages and accurately elucidated the 3D structure of seminiferous tubules, including the number and length of tubules as well as the numbers of connections with the rete testis, branching points, and blind ends. We also developed a technique to identify the precise spermatogenic stage and cellular composition of the seminiferous epithelium. It consists of the combination of lectin histochemistry for acrosomes and immunohistochemistry for specific cell markers visualized with fluorescence. Using this method, we examined seminiferous tubules from normal mice and counted the number of each cell type at each stage, and thereby established a quantitative standard for the cellular composition of the seminiferous epithelium. We then investigated seminiferous epithelia from genetically modified infertile mice deficient in certain cell adhesion molecules and revealed characteristic abnormalities in the cellular composition. We also analyzed the distribution and direction of spermatogenic waves along the length of adult seminiferous tubules as well as the site of the first onset of spermatogenesis in postnatal seminiferous tubules. These methods will be useful for investigating the structure and function of seminiferous tubules in mice and humans under normal and pathological conditions.

Ductuli efferentes of the male Golden Syrian hamster reproductive tract.

Efferent ductules are responsible for the transportation of spermatozoa from the testis to the epididymis and their epithelium is responsible for the reabsorption of over 90% of the luminal fluid. The purpose of this research was to characterize the gross morphology and histology of efferent ductules in the male Golden Syrian hamster. The efferent ductules emerge from rete testis with a unique polarity at the apex or cephalic pole of the testis. The number of efferent ductules varied from 3 to 10 with an average of 6.0 and blind ending ducts were observed in approximately 56% of the males. The ductules merged into a single common duct prior to entering the caput epididymidis. The proximal efferent ductule lumen was wider than the distal (conus and common ducts), consistent with reabsorption of most of the luminal fluid, as was morphology of the ductal epithelium. Non-ciliated cells in the proximal region had prominent endocytic apparatuses, showing both coated pits and apical tubules in the apical cytoplasm. Large basolateral, intercellular spaces were also present in the epithelium of the proximal region. Distal non-ciliated cells had an abundance of large endosomes and lysosomal granules. Localisation of sodium/hydrogen exchanger-3 (NHE3; SLC9A3) and aquaporins 1 and 9 (AQP1, AQP9) along the microvillus border was also consistent with ion transport and fluid reabsorption by this epithelium. In comparison, the caput epididymidis epithelium expressed only AQP9 immunostaining. Another unusual feature of the hamster efferent ductules was the presence of glycogen aggregates in the basal cytoplasm of small groups of epithelial cells, but only in the proximal ducts near the rete testis. Androgen (AR), estrogen (ESR1 and ESR2) and vitamin D receptors (VDR) were also abundant in epithelial nuclei of proximal and distal efferent ductules. In comparison, caput epididymidis showed very little immunostaining for ESR1.

Variant left testicular artery from the superior polar artery and triple right renal arteries: a case report

Testicular arteries are the branches of the abdominal aorta below the level of origin of renal arteries. They may originate at a higher or a lower level from the aorta. However, when they originate from some other artery, they become surgically significant, since the ligature of the main artery giving rise to the testicular artery might lead to testicular atrophy. The present report is about the origin of the left testicular artery from the superior polar artery, which is a branch directly from the abdominal aorta above the renal artery. With the advent of novel surgical techniques, prior knowledge of rare variation in the testicular arteries becomes significantly important during surgery for renal transplant procedures, as well as for undescended testis or varicocele (AU)

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Proximal testicular ducts of the Mediterranean gecko (Hemidactylus turcicus).

The efferent ducts of the Mediterranean Gecko, Hemidactylus turcicus (Gekkonidae) were investigated using light and electron microscopy. The seminiferous tubules unite into a single rete testis tubule. The rete testis divides into 3-4 ductuli efferentes which all drain into the cranial portion of the ductus epididymis. All efferent ducts are most active during the months of December to August. The rete testis is composed of a simple squamous epithelium with bifurcated nuclei and a labyrinthine network of intercellular canaliculi. Ciliated and nonciliated cells are present, and more than one cilium extends from the scattered ciliated cells. The presence of small clear vesicles and widened intercellular canaliculi suggest that cells of the rete testis are responsible for intake of luminal fluids. The ductuli efferentes are composed of a simple cuboidal epithelium consisting of ciliated and nonciliated cells, and ciliated cells are the dominant cell type. During the inactive season the number of lysosomes increases and the cells become spermiophagic. The ductus epididymis is composed of a tall pseudostratified columnar epithelium with relatively scarce basal cells. No evidence for regionalization was observed. The ductus epididymis is highly secretory during the active season with numerous electron-dense secretory granules, whose glycoprotein products are released by merocrine secretion. Basally, the active epididymis has swollen intercellular canaliculi and enlarged cisternae of rough endoplasmic reticulum. During the inactive season the secretory activity decreases and membranous structures and fibrous material are observed within widened intercellular canaliculi apical to the basal cells.

Absorptive activities of the efferent ducts evaluated by the immunolocalization of aquaporin water channels and lectin histochemistry in adult cats.

Ultrastructural and biochemical features of efferent ducts (EDs) are indicative of an intense absorptive activity towards the luminal fluid. This function was investigated by 1) the immunohistochemical localization of different aquaporins, integral membrane water channels that facilitate rapid passive movement of water, and 2) the histochemical localization of lectins, known to have specific affinity for glycoconjugate residues. AQP1 was mostly revealed at the apical surface and adluminal cytoplasm of non-ciliated cells and to a minor extent in their lateral plasma membrane, whereas it was absent in ciliated cells. Blood vessels showed AQP1-immunoreactivity, which was present in endothelial cells of venous vessels and capillaries and around the muscular sheath of arteries. AQP9 was expressed in the apical zone of ciliated and non-ciliated cells and in the lateral cell membrane. AQP2 and AQP5 were undetectable. Lectin histochemistry showed that non-ciliated cells contain glycans with terminal Neu5Acalpha2,3Galbeta1,3GalNAc, Neu5Acalpha2,3Galbeta1,4GlcNAc, Galbeta1,4GlcNAc, GalNAc (s-PNA, MAL II, RCA120, SBA reactivity) and with internal/terminal alphaMan (Con A affinity) at the luminal surface and the apical region. In addition, non-ciliated cells expressed oligosaccharides terminating with GalNAc and Neu5Acalpha2,6Gal/GalNAc (SNA reactivity) in the luminal surface and the apical zone, respectively. Ciliated cells revealed glycoconjugates only on cilia, which showed terminal Neu5Acalpha2,3Galbeta1,4GlcNAc (s-RCA120 staining) and GalNAc, as well as internal/terminal alphaMan and GlcNAc (s-WGA, GSA II staining). Data provide evidence for the involvement of different pathways in the bulk reabsorption of water and low molecular weight solutes by the non-ciliated cell of the cat EDs. AQP-mediated trans-cellular route can be hypothesized, together with fluid phase endocytosis mediated by the glycocalix and a well-developed endocytotic apparatus. Epithelial ciliated cells, whose main function is the movement of luminal content, might also participate in absorptive processes to a lesser extent.

Observações morfológicas no epitélio de revestimento da rede testicular (RT) de codorna (Coturnix coturnix) da variedade italiana / Morphological features on the surface epithelium of the rete testis (RT) of quail (Cotunix coturnix) from the italian variety

A rede testicular (RT) de codorna é predominantemente do tipo intra-albugínica e constituída por canais tortuosos, revestidos por células epiteliais, estendendo-se pela túnica albugínea do testículo e a seguir penetram na estrutura microscópica da região epididimária (RE), pelo seu tecido matricial. Os canais da RT são contínuos com os ductos eferentes proximais da RE. A ultraestrutura do epitélio de revestimento da RT mostra diferenças marcantes na primavera - a fase proliferativa do ciclo reprodutivo, e em meados do outono - a fase quiescente do ciclo reprodutivo nesta ave. As características ultraestruturais marcantes dos epiteliócitos da RT, nestas duas fases, foram a baixa eletrodensidade citoplasmática e ocorrência de poucas vesículas e lisossomos no outono, que aparecem com maior freqüência na RT primaveril. Aspectos degenerativos de organelas, e presença de extrusões citoplasmáticas apicais, apareceram em número expressivo no outono, não se observando aspectos ultraestruturais marcantes nas estações de inverno e verão, relativamente às observações de primavera.

The RT of domestic quail from Italian variety showed mainly an albuginic pattern being represented by tortuous channels lined predominately by a simple cubic epithelium. RT channels extended along the testicular albuginea and penetrates into the epididymal region (ER) through its myoconnective matrix. Passageways were continuous to proximal efferent ducts of the ER. Epithelium lining ultrastructure of RT passageways showed some differences between the spring and the inactive phase at middle fall, concerning the quail testicular reproductive cycle. The features observed in RT epitheliocytes in fall were the low cytoplasmic electrodensity, paucity of supranuclear vesicles, which were abundant and variable in form and shape in spring, and some degenerative aspects of cell organelles mainly in ER lamellae. Moreover, presence of apical cytoplasmic extrusions were verified in the fall. No marked features were seen in the RT ultrastructure during the winter and summer comparatively to the active phase of spring.

Three-dimensional visualization of testis cord morphogenesis, a novel tubulogenic mechanism in development.

Testis cords are specialized tubes essential for generation and export of sperm, yet the mechanisms directing their formation, and the regulation of their position, size, shape, and number remain unclear. Here, we use a novel fluorescence-based three-dimensional modeling approach to show that cords initially form as a network of irregular cell clusters that are subsequently remodeled to form regular parallel loops, joined by a flattened plexus at the mesonephric side. Variation in cord number and structure demonstrates that cord specification is not stereotypic, although cord alignment and diameter becomes relatively consistent, implicating compensatory growth mechanisms. Branched, fused, and internalized cords were commonly observed. We conclude that the tubule-like structure of testis cords arise through a novel form of morphogenesis consisting of coalescence, partitioning, and remodeling. The methods we describe are applicable to investigating defects in testis cord development in mouse models, and more broadly, studying morphogenesis of other tissues.

Variabilidade sazonal no ducto epididimário de codorna doméstica: observações morfológicas / Seasonal variability in epididymal duct of the domestic quail: morphologic features

O ducto epididimário (DE) de codorna doméstica mostrou, ao longo do ano, variabilidade pequena, porém muito expressiva no outono, o qual corresponde à fase quiescente do ciclo testicular anual. A morfologia do DE na primavera foi, em termos, similar à verificada no verão e inverno. Nestas fases notaram-se aumento significante do calibre tubular do DE; estocagem intraluminal de espermatozóides e ocorrência de mitocôndrias, lamelas do RE, vesículas variáveis quanto à forma, dimensões e conteúdos e presença de alguns lisossomos localizados, principalmente, no citoplasma apical das células principais (P), no epitélio epididimário. Estas características ultra-estruturais das células P parecem ser indicativas da ocorrência de processos ativos de endocitose e de secreção micromerócrina. A quiescência outonal foi caracterizada pelo aspecto anfractuoso do DE; ausência de espermatozóides e pouco material intraluminal, observados à microscopia de luz. Características ultra-estruturais degenerativas foram verificadas ao nível do citoplasma supranuclear das células P epididimárias no outono.

Small but expressive variability was noted on the epididymidis duct (ED) of domestic quail along the year, with more evidence in autumn of the quiescent phase of the annual testis cycle in this species. Spring features of ED had a general similar pattern in summer and winter. They were characterized by enlargement of epididymis tubule, storage of spermatozoa into the luminal compartment and presence of mitochondria, ER lamellae, several variable vesicles, and lysosomes localized mainly on the apical cytoplasm of principal cells (P) of the epididymal epithelium. These P cells features indicated a process of endocytosis and perhaps protein secretion. Autumn quiescence was marked by a convolute pattern of the epididymis tubule, lacking of spermatozoa and small amount of exfoliate heterogeneous material inside the luminal compartment at light microscopy. Ultrastructural degenerative features mainly apical cytoplasmic debris were seen in the supranuclear cytoplasm of lining P cells.

Distribution of vitamin D3 receptor in the epididymal region of roosters (Gallus domesticus) is cell and segment specific.

Vitamin D3 is a steroid hormone well known by its role in maintaining calcium homeostasis, however this hormone may also participate in other biological functions, including control of reproductive processes. The vitamin D3 action is mediated by the vitamin D3 receptor (VDR). VDR is widely distributed in the rodent reproductive tract, however the occurrence of VDR and the role of the vitamin D3 in the avian reproductive tract remain unknown. The aim of the present study was to investigate the expression and cellular distribution of VDR in the epididymal region of roosters. VDR expression was investigated by Western blotting analysis and the tissue distribution of the receptor was determined by immunohistochemistry. The Western blotting assay revealed a major VDR protein band of 61kDa in the epididymal region of rooster. Nuclear VDR expression was found in all segments of the epididymal region, namely rete testis, efferent ductules, connecting ducts and epididymal ducts. Nonciliated cells of the distal efferent ductules showed the highest levels of VDR expression, followed by the proximal efferent ductules and rete testis. The connecting and epididymal ducts showed less intense VDR immunostaining. The differential VDR expression in the epididymal region segments reveals that several extratesticular ducts may be target for vitamin D3 action and suggests that vitamin D3 may have a regional-specific function, such as calcium transport, that is modulated through VDR activity.

Excurrent duct system of the male turtle Chrysemys picta.

The epididymis and efferent duct system of the turtle Chrysemys picta were examined. Seminiferous tubules are drained by a series of ducts that form a rete exterior to the tunica albuginea. The rete is located lateral to the testis and consists of anastamosing tubules of varying diameters, lined by a simple epithelium consisting of squamous to cuboidal cells. The rete is highly vascularized. A series of tubules (efferent ductules) connect the rete to the epididymis proper. The efferent ductules are highly convoluted, running between the epididymal tubules and are of varying diameters. The simple columnar epithelium lining these tubules possesses tight junctions, with every third or fourth cell possessing long cilia that protrude into the lumen. The cytoplasm of these epithelial cells contains abundant mitochondria. In the central portion of the efferent ductule, epithelial cells possess granules that appear to be secreted into the lumen by an apocrine process. The epididymis proper is a single, long, highly convoluted tubule that receives efferent ductules along its entire length. It is lined by a pseudostratified epithelium containing several cell types. The most abundant cell (vesicular cell) lacks cilia, but has a darkly staining apical border due to numerous small vesicles immediately beneath the luminal membrane. The small vesicles appear to fuse with each other basally to form larger vesicles. These cells appear to have an absorptive function, and occasionally sperm are embedded in their cytoplasm. The second-most abundant cell is a basal cell found along the basement membrane. The number of these cells fluctuates throughout the year, being most abundant in late summer and early fall. A small narrow cell with an oval nucleus and darkly staining cytoplasm, extending from the basement membrane to the apical surface, is present in small numbers, particularly in the caudal regions of the epididymis. This cell is frequently found in association with another narrow cell having a rounded nucleus and abundant mitochondria in its cytoplasm.

Cyclical reproductive changes in the non-ciliated epithelia of the epididymis of birds.

The epididymis of two species of domestic birds, the domestic fowl (Gallus domesticus), duck (Anas platyrhynchos), and of domestic and feral guinea-fowl (Numida meleagris) was studied during the three main phases of the reproductive cycle (prepuberal, sexually mature and active, and sexually mature but inactive or resting) with a view to identifying major histological and ultrastructural changes associated with and distinctive for each phase. Rete testis cells accumulated numerous variably sized lipid droplets in all birds, as well as large heterogeneous and lipofuscin-containing dense bodies in the guinea-fowl, during the resting but not in the other phases. The principal or Type III cells of the connecting and epididymal ducts exhibited profound structural changes, including, but not limited to, rarefied cytoplasm, inconspicuous and general loss of sparsely granular endoplasmic reticulum, loss of secretory vesicles in the drake, and an enhanced and conspicuous presence of lipid droplets in the guinea-fowl. The rete cells appeared to be less sensitive than the Type III cells to a reduced level or absence of lumenal androgens. These phase-dependent changes may help to prevent or minimize discrepancies in the interpretation of the normal structure of the epididymis in birds during the sexually active phase, as distinct from the other two phases and their intermediate phases.

Pictorial review: ultrasound appearances of the rete testis.

The rete testis is formed in the fifth week of intra-uterine life. The epithelium along the medial side of the mesonephric ridge thickens to form a genital ridge; cellular gonadal cords at the periphery of the ridge unite with a tubular network from the mesonephric mesenchyme forming the testicular rete. On ultrasound, a range of normal appearances of the rete testis is recognised from ill-defined areas of decreased reflectivity to a coarse tubular appearance (often with finger like projections). In the present review the embryological development of the rete testis is briefly outlined. In addition, the important anatomical variations, pertinent to ultrasound imaging, will be presented.

A rede testicular na cobaia: observaçöes morfológicas e histoquímicas / The rete testis in guinea pig: a morphological and histochemical examination

A rede testicular (RT) da cobaia tem características morfológicas de um tipo intermediário de RT. Esta rede foi caracterizada com cavitária, labiríntica e axial relativamente à disposiçäo de seus canais epiteliais interconectados. Morfologicamente consiste, predominantemente, de câmaras epiteliais interligadas que penetram no parênquima testicular, por uma pequena distância. Os canais e câmaras de todas as partes da rede säo revestidos por epitélio cúbico simples. Reatividades histoenzimáticas fortes foram observadas, predominantemente, na matriz da RT às enzimas fosfatases alcalina e ácida e m-ATPase. Ambas as fosfatases mostraram reatividades médias no epitélio da RT de cobaia, no qual a reatividade da m-ATPase foi fraca. Por outro lado, a NADH-d mostrou reatividade média, porém homogênea, na matriz e no epitélio da RT.

Contribuiçäo ao estudo do funículo espermático no morcego (Phyllostomus hastatus) / Contribution to the study of spermatic cord in bat (Phyllostomus hastatus)

Neste trabalho sao descritas observaçoes anatômicas sobre o suprimento arterial e drenagem venosa testiculares no morcego Phyllostomus hastatus que tem hábito alimentar misto (frutas, insetos e pequenos vertebrados). As disposiçoes e comportamento anatômico dos vasos testiculares arteriais e venosos, incluindo aspectos histológicos, na intimidade do funículo espermático sao comparadas com descriçoes em outras espécies de mamíferos.

Reexamination of the morphology of the extratesticular rete and ductuli efferentes in the goat.

The morphology of the extratesticular rete and ductuli efferentes was reexamined in serial cross sections collected from the entire mass of the efferent ductules and in longitudinal sections collected from the partially unraveled efferent ductules. The extratesticular rete forms a 3-4-mm-long sac-like dilatation, which, within the head of the epididymis, has a wide lumen (up to 4 mm) and gives off along its length numerous evaginations, which, in turn, make connections with the ductuli efferentes. The latter is a mass of 16-18 ductules lined by three types of nonciliated cells: type II cells are characterized by dense, periodic acid-Schiff (PAS)-positive granules; type III cells are characterized by empty-appearing, PAS-negative vacuoles; and type I cells lack both granules and vacuoles. The distribution of the three types of nonciliated cells varies along the length. Whereas only type I cells are present in the beginning portion of the efferent ductule, type II cells predominate in the middle portion and type III cells in the distal portion (near the epididymis). The transition from one cell type to the other type is gradual; thus there are short segments along the length that share characteristics first for type I and type II cells and then for type II and type III cells. These results demonstrate that different nonciliated cell types are not randomly distributed in the epithelium of the ductuli efferentes but, instead, gradually differentiate from type I to type II to type III cells along the length of each efferent ductule. Factors controlling this differentiation remain to be studied.

Blind-ending tubules and branching patterns of the rat ductuli efferentes.

The ductuli efferentes of Sprague-Dawley rats were studied by microdissection and microscopic evaluation to document the presence of blind-ending tubules (ductuli aberrantes) and to describe morphological and ultrastructural differences between normally open ductules and blind-ending tubules. The branching patterns of the ductuli efferentes varied considerably between animals. A majority of the animals studied had either six or seven ductuli connected to the rete testis, with some animals having as few as four or as many as eight. Pairs of ductules began merging in the conus vasculosa, ultimately forming a single terminal duct within the capsule of the initial segment epididymidis. In a majority of animals, the junctions were unequally matched and located at various positions within the conus. Blind-ending tubules, found in 60% of the animals, were surrounded by thick connective tissue, and had a smaller diameter (78.7 +/- 1.4 microns) than normal ductules in the conus vasculosa (119.5 +/- 2.1 microns) or the terminus (102.2 +/- 1.5 microns). The lumina of blind-ending tubules were contracted and did not contain sperm. Nonciliated cells in the epithelium of blind-ending tubules contained fewer PAS-positive granules and electron-dense bodies (lysosomes) than nonciliated cells in normal ductules. Consideration of these characteristics will prevent blind-ending tubules from being mistaken for pathological changes in efferent ductules.

[Structure of the rete testis of the cat (Felis domestica, L)]. / Struktur des Hodennetzes der Katze (Felis domestica, L.).

The rete testis of the cat consists of 3 parts: a septal or interlobular part; a mediastinal part and a tunical part. The septal part contains the septal or transitory tubuli recti and the tubuli recti. The transitory tubules are formed as a confluence of the seminiferous tubules at the apex of the testicular lobules and the tubuli recti. The mediastinal rete is formed of long, straight channels which increase in size and become more irregular and anastomotic below the tunica albuginea at the cranial extremity of the testis. The end is characterized as the tunical part of the rete testis and communicates with the extratesticular rete testis. The channels all parts of the rete are lined by simple cuboidal or columnar epithelium. These epithelial channels are supported by a connective tissue containing smooth muscle cells. The framework tissue of the rete is more conspicuous at the cranial extremity of the testis, with a mio-connective matrix organization.

A morphological study of the efferent ducts of the human epididymis.

Efferent ducts of the human epididymis were studied with a light microscope and a computerized three-dimensional image analyser by preparing complete serial sections. The efferent ducts were characterized by a columnar epithelium, which differed both from the cuboidal epithelium of the rete testis and from the tall columnar epithelium of the epididymal duct. Therefore, the junctions of the efferent ducts with the rete testis and epididymal duct could be identified morphologically. Five or six efferent ducts originated from the dilated extra-testicular rete testis, ran in an extremely tortuous manner and transformed into the epididymal ducts in an end-to-end pattern near the border of the epididymal head and corpus. Computerized image analysis confirmed light microscopical findings and demonstrated three-dimensional structures of the junctions of the efferent ducts with both the rete testis and the epididymal ducts.

Observaçöes sobre a histoarquitetura geral dos testículos do hamster pós-natal / Observations on the general histoarchitecture of the post-natal hamster's testes

O presente trabalho é uma descriçäo sobre a morfologia da cápsula testicular e do complexo da rede testicular no hamster da variedade champanha, entre 10 e 90 dias de vida pós-natal. Adicionalmente, säo descritas algumas peculiaridades morfológicas dos túbulos seminíferos e do epitélio seminífero. Outras observaçöes apresentadas sobre a morfologia dos túbulos seminíferos e superficial da rede testicular