Three-dimensional analysis and in vivo imaging for sperm release and transport in the murine seminiferous tubule.

Spermatozoa released from Sertoli cells must be transported to the epididymis. However, the mechanism of the luminal flow in seminiferous tubules has remained unclear to date. Therefore, in this study, we investigated luminal flow and movements in the seminiferous tubules by three-dimensional analysis and in vivo imaging. Serial 5-µm-thick mouse testicular sections at 50-µm-intervals were prepared and stained by Periodic Acid-Schiff-hematoxylin. After three-dimensional reconstruction of the seminiferous tubules, the localization of the released spermatozoa and the stages observed in the sections were recorded in each reconstructed tubule. Luminal movements in the seminiferous tubules were observed by in vivo imaging using a fluorescent-reporter mouse and two-photon excitation microscopy system. Spermatozoa without contact to the seminiferous epithelium were not accumulated toward the rete testis. Additionally, such spermatozoa were found on their way not only to the most proximal rete testis but also a more distant rete testis from any stage VIII seminiferous epithelia. In vivo imaging demonstrated that the direction of the flagella of spermatozoa attached to the seminiferous epithelium was repeatedly reversed. The epithelium at the inner curve of the seminiferous tubule was shaken more actively and had fewer spermatozoa attached compared with the epithelium at the outer curve. Our results hence suggest that the luminal flow in the seminiferous tubules is repeatedly reversed and that this physical force helps spermatozoa to be released from Sertoli cells. In brief: Spermatozoa are released from Sertoli cells and flow in the seminiferous tubule to the rete testis. Our results suggest that the luminal flow in the tubules is repeatedly reversed and that this physical force helps spermatozoa release from the Sertoli cells.

Primary cilia: biosensors of the male reproductive tract.

BACKGROUND: The primary cilium is a microtubule-based organelle that extends transiently from the apical cell surface to act as a sensory antenna. Initially viewed as a cellular appendage of obscure significance, the primary cilium is now acknowledged as a key coordinator of signaling pathways during development and in tissue homeostasis. OBJECTIVES: The aim of this review was to present the structure and function of this overlooked organelle,with an emphasis on its epididymal context and contribution to male infertility issues. MATERIALS AND METHODS: A systematic review has been performed in order to include main references relevant to the aforementioned topic. RESULTS: Increasing evidence demonstrates that primary cilia dysfunctions are associated with impaired male reproductive system development and male infertility issues. DISCUSSION: While a large amount of data exists regarding the role of primary cilia in most organs and tissues, few studies investigated the contribution of these organelles to male reproductive tract development and homeostasis. CONCLUSION: Functional studies of primary cilia constitute an emergent and exciting new area in reproductive biology research.

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.

Need based structural changes in the ductulus efferentis during sperm movements in the human epididymis.

The dynamic structures of the human ductuli efferentes, which connect between the rete testis and the ductulus epididymidis, are described in the present paper. Maturation of sperm takes place in the tubule and after this process the group of sperm is transferred to the ductus. All ductuli do not show the same histological contents. Each ductulus shows variety of different configuration depending on the functional phase (the state of sperm maturation) of the tubule. The basic pattern of all the tubuli is the same, however, they differ in the thickness, cytoplasmic components of epithelial cells and luminal contents. Larger tubule has big lumen whereas small is almost without a lumen. During the process, spherical and luminal macrophages act as nurse macrophage by using G1 granules obtained from the epithelial cell, and irregular shaped macrophage in the small tubule acts as scavenger macrophage. It has been shown that the epithelial cell of the large tubule show intense excretory activity and the secretory substance is different from other granules and lysosomes by periodic acid methenamine silver staining.

The ultrastructure of the guinea pig rete testis.

The chambers of the rete testis (RT) of guinea pig are lined by a simple epithelium, whose cells are squamous, cubical and columnar in shape. The epithelial cells with distinct shapes were counted and the quantitative analysis of the number of these cells showed relative predominance of cubical cells. The ultrastructural observations showed predominance of membrane interdigitations among the epithelial cells. These cells present common cytoplasmic organelles. The Golgi complex polarity is typical with observation of electronlucent vesicles on the Golgi cis face closely related to rough endoplasmic reticulum (ER) lamellae, mitochondria and large number of polysomes on the Golgi trans face. These related structures present in Golgi area of RT cells suggest secretory activity which maybe occurs in the RT epithelium. Endocytotic process also occurs in the RT and this function probably concerns the uptake of substances and resorption of seminiferous fluid. Apical cilia present in RT epithelium cells are related with fluid transport and perhaps with chemoreception. Presence of spermatozoa portions enclosed into the cytoplasm of some epithelium cells has been referred to as spermatophagy. The RT complex is mainly supported by loose connective tissue, with collagen fibres and some Leydig cells. Leydig cells are adjacent to the network channels of the septal part of the RT and apparently are able to secrete inside the RT lumen.

The ductuli efferentes testis of the greater cane rat ( Thryonomys swinderianus).

The structure of the efferent ducts of animals is known to vary from one species to another, it even varies between segments of the ducts in the same species. In the grasscutter or greater cane rat ( Thryonomys swinderianus), there are three segments of the efferent duct, based on their content of non-ciliated or principal cell types. Type I non-ciliated cell is present exclusively in the long proximal part of the duct, and exhibits a well-developed subapical endocytic apparatus as well as numerous oval or pleomorphic dense bodies. The type II non-ciliated cell predominates in the middle part of the duct, displays a poorly developed subapical endocytic apparatus but contains large, numerous vacuoles and dense bodies, all of which fill about two-thirds of the cell height. The type III non-ciliated cell, found in the epithelium of the terminal part of the duct, is poorly endowed with a subapical endocytic apparatus and contains no conspicuous endocytic vesicles or vacuoles. Only a few, small, dense bodies are present, if at all. The efferent duct of the cane rat is thus similar to that of man, the bull, goat and dog, in containing three varieties or types of non-ciliated cells. This report is the first to describe multiple non-ciliated cells in the epithelium of the efferent ducts of a rodent and, indeed, of a mammal smaller than the dog.

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.

Blockage of the rete testis and efferent ductules by ectopic Sertoli and Leydig cells causes infertility in Dax1-deficient male mice.

DAX-1, an X-linked member of the orphan nuclear receptor superfamily of transcription factors, plays a key role in sex determination and gonadal differentiation. Dax1-deficient male mice are infertile and have small testes despite normal serum levels of T and gonadotropins. Examination of Dax1-deficient testes reveals dilated seminiferous tubules and abnormal parameters of sperm fertilizing capability consistent with a possible obstruction in the testis. To test this hypothesis, we performed a comprehensive evaluation of the male reproductive tract in Dax1-deficient mice. Light and electron microscopic examination revealed the rete testis is blocked by aberrantly located Sertoli cells, creating a tailback of necrosing sperm in the testis. Sertoli cells also obstruct the proximal and middle efferent ductules, and this is accompanied by an overgrowth of the efferent duct epithelium. Seminiferous tubules close to the rete testis contain ectopic Leydig cells, distinct from the hyperplastic Leydig cells present in the interstitial space. The peritubular tissue surrounding these tubules is frequently abnormal, containing relatively undifferentiated myoid cells and no basement membrane between the myoid cells and Sertoli cells. A third of aged (>1-yr-old) Dax1-deficient male mice develop sex cord-stromal tumors, derived from cells of the Sertoli/granulosa cell or Leydig cell lineages. Combined, these observations reveal abnormal differentiation and proliferation of Leydig cells and Sertoli cells in Dax1-deficient male mice, leading to obstruction of the rete testis and infertility.

Estrogen receptor alpha has a functional role in the mouse rete testis and efferent ductules.

Previous studies of the estrogen receptor-alpha knockout (alpha ERKO) in the male mouse demonstrate that the rete testis and efferent ductules are targets of estrogen. Because the alpha ERKO mouse lacks a functional estrogen receptor alpha (ER alpha) throughout development, it was not known whether the morphological and physiological abnormalities observed in the alpha ERKO male were due to developmental defects or to dysfunctions concurrent with the lack of ER alpha in the tissue. This study was designed to determine if treatment of normal wild-type (WT) mice with the pure antiestrogen, ICI 182,780, (ICI) could reproduce the morphological characteristics seen in alpha ERKO mice. Thirty-day-old male mice were treated for 35 days with either castor oil or ICI. Age-equivalent alpha ERKO mice were used for comparison. Light microscopic examinations of the reproductive tracts revealed dramatic changes in the efferent ductules of treated mice: a 1.7-fold increase in luminal diameter, a 56% reduction in epithelial cell height, a 60% reduction in brush boarder height of nonciliated cells, and an apparent reduction of the number of observable lysosomes and endocytotic vesicles. Testes of ICI-treated mice showed swollen rete testes area (6.5 times larger than control) and a 65% reduction in rete testis epithelium height. However, there were no significant changes in body and testis weights. These results indicate that ER blockage with ICI in WT mice results in morphological changes of the efferent ductules resembling those seen in alpha ERKO siblings of the same age. Based on this study, we conclude that ER alpha has a functional role in the mouse reproductive tract and the aberrant morphology observed in the efferent ductules of the alpha ERKO mouse is likely the result of a concurrent response to the lack of functional ER alpha, and not solely due to the lack of ER alpha during early developmental times.

Postnatal differentiation of efferent ductule epithelium in goats: a light microscopic and ultrastructural study.

Caprine efferent ductule epithelium contains ciliated and nonciliated cells. The latter cells are divided into three types: type II cells contain PAS-positive granules, type III cells contain PAS-negative vacuoles, and type I cells lack both granules and vacuoles (Goyal and Williams, Anat. Rec. 220:58-67). The objectives of this study are i) to determine when the epithelium differentiates into ciliated and nonciliated cells, ii) to determine when nonciliated cells acquire characteristics typical for type II and type III cells, and iii) to relate developmental changes in the epithelium with those in the testis. Testes and efferent ductules were examined at the light and electron microscopic levels in goats from 1-25 weeks of age. Efferent ductule epithelium contained ciliated and nonciliated cells as early as week 1. While ciliated cells were differentiated at week 1, differentiation of nonciliated cells did not occur until week > or =15. Differential features in ciliated cells included the presence of cilia at the apical border and an aggregation of mitochondria in the apical cytoplasm. Those in nonciliated cells included the presence of i) an endocytotic apparatus at week > or =15, ii) PAS-positive granules at week > or =15, and iii) PAS-negative vacuoles at week > or =25. The seminiferous tubules developed lumens at 12-15 weeks. Hence, while differentiation of ciliated cells occurred much before lumen formation in the seminiferous tubules, that of nonciliated cells coincided with, or occurred soon after, lumen formation, suggesting a role for testicular fluid contents in their differentiation. The goat efferent ductules can be characterized morphologically mature by 25 weeks.

Micropuncture and cannulation studies of fluid composition and transport in the ductuli efferentes testis of the rat: comparisons with the homologous metanephric proximal tubule.

Luminal fluids were collected in vivo by micropuncture and cannulation from the rete testis, efferent ducts and ductus epididymidis of the rat to determine the composition of efferent duct fluids and the rates of reabsorption of water and solutes by the efferent ducts. The concentration of spermatozoa increased by a factor of about 25 from 2.42 x 10(4) microliters-1 in the fluid from the rete testis to 6.00 x 10(5) microliters-1 in fluid at the end of the efferent ducts, indicating that 96.2% of the fluid leaving the testis is reabsorbed from the lumen of the efferent ducts. Most of this reabsorption (70.9% or 33.4 microliters h-1) occurs in the region between the rete testis and the middle of the coni vasculosi, with only 25.1% (11.8 microliters h-1) occurring between the coni and the beginning of the ductus epididymidis. However, reabsorption across the epithelium occurs at about the same rate in both regions, with the proximal region reabsorbing 17.2 microliters cm-2 h-1 (70.9% of fluid entering the region) and the distal region reabsorbing 12.2 microliters cm-2 h-1 (86.1% of fluid entering the region). Consequently, the fluid reabsorption rate for the whole efferent duct system (15.6 microliters cm-2 h-1) is similar to the values for individual regions. The principal solutes in luminal fluids from the efferent ducts are Na+ (137-144 mM) and Cl- (113-130 mM). The estimated sum contribution of Na+, Cl- and K+ to the osmotic pressure of luminal fluids was approximately 80% at each site sampled in the efferent ducts. The osmotic pressure of luminal fluid samples (301-307 mosmol kg-1) did not vary significantly along the ducts or differ significantly from that of blood plasma. The results demonstrate that there is a net reabsorption in the efferent ducts of nearly all the testicular output of water and inorganic electrolytes, and most of the protein, and that, in comparison, the ductus epididymidis is a negligible site of net fluid reabsorption. The results indicate that the ductus epididymidis, rather than the efferent ducts, is the site of accumulation of high concentrations of specific organic compounds like inositol. The efferent ducts are similar to the homologous proximal tubules of the metanephric kidney in that the luminal electrolyte composition (principal solutes Na+ and Cl-) and osmotic pressure remain relatively stable and that fluid reabsorption is close to isotonic and occurs at the same rate as the reabsorption of Na+.

Central effect of rete testis fluid, inhibin 32K, and follicular fluid on plasma gonadotropin concentrations in sheep: inhibin is not the rete testis fluid protein able to suppress luteinizing hormone pulses.

We have previously shown that peripheral administration of rete testis fluid (RTF) proteins was able to suppress LH pulses through the suppression of LHRH pulses. This activity was named "LHRH Statin." The aims of the present work were to analyze LH inhibition after an intracerebroventricular injection of RTF and to determine whether inhibin is the factor responsible for this inhibition. Castrated rams (experiment 1) or ewes (experiment 2) received an intracerebroventricular injection of RTF, purified bovine inhibin 32K, bovine follicular fluid, or human serum albumin as control. Animals were bled every 15 min for 5 h before injection and for 7 h after injection. LH mean levels were significantly lowered (p < 0.01) only in the RFT-treated groups. FSH levels were not affected irrespective of group, source, or dose of inhibin. These experiments show first, that protein(s) present in ovine RTF can suppress LH secretion in sheep; second, that bovine follicular fluid or purified bovine inhibin 32K have no effect on LH secretion. Furthermore, the results suggest that centrally administered inhibin has no effect on FSH secretion under our experimental conditions. Together, these experiments clearly demonstrate that inhibin 32K does not exert any "LHRH statin" activity.

Comparisons of endocrinological and testis parameters in 18-month-old Ile de France and Romanov rams.

Endocrinological and testis parameters of adult 18-month-old Ile de France (IF) and Romanov (Ro) rams were compared during sexual season. Testis weights, total volumes of intertubular tissue, and of blood and lymph vessels, total seminiferous tubule length, rete testis flow rate and daily production of germ cells were significantly higher in IF than in Ro rams. These variations originated from differences in Sertoli cell numbers, which were established before puberty. When daily productions of germ cells, of ABP or of RTF were expressed per Sertoli cell, they were higher in Ro than in IF rams. Quality of spermatids, as measured by their cellular size prior to elongation, was lower in Ro than in IF. The number of FSH-binding sites per Sertoli did not differ between the two breeds but FSH plasma levels were higher in Ro than in IF rams. Total numbers of Leydig cells per testis, their individual size or their LH-binding capacity did not differ significantly between the two breeds. However, the ratio of mean testosterone upon mean LH plasma levels were greater in Ro than in IF rams while both breeds had identical LH mean plasma levels.

Influence of rete testis fluid deprivation on the kinetic parameters of goat epididymal 5 alpha-reductase.

A surgical technique to cannulate the rete testis of the goat was utilized to examine the effects of rete testis fluid (RTF) deprivation on the enzymatic activity of epididymal 5 alpha-reductase. Kinetic techniques were used to determine whether the regional enzymatic effect of RTF deprivation is to decrease the apparent number of 5 alpha-reductase active sites or the catalytic activity of each active site within the epididymal epithelium. Paired comparisons of (Vmax)app and (Km)app values between control and RTF-deprived epididymides indicated that RTF deprivation affected the value of (Vmax)app with no apparent change in the values of (Km)app in caput, corpus, and cauda epididymal regions. We conclude that RTF deprivation in the goat epididymis for 7 days results in a decreased number of apparent 5 alpha-reductase active sites within the epididymal epithelium.

Effect of deprival of rete testis fluid on the morphology of efferent ductules.

One week after unilateral cannulation of the rete testis and ligation of the efferent ductules, samples of the proximal, middle and distal segments of the efferent ductules of 6 goats were examined by light and electron microscopy and compared with normal contralateral efferent ductules. The pseudostratified columnar epithelium consisted of ciliated, nonciliated and basal cells. The number of clear vacuoles decreased markedly in the proximal and middle segments following deprivation of androgen-rich rete testis fluid. The epithelium of the distal segment of the cannulated side had few large clear vacuoles compared to the normal side, which had a high concentration of large vacuoles. Since the large vacuoles decreased in all three segments following ligation, they were thought to be absorptive. Some cells of the distal segment of the cannulated side contained a single, huge, basal vacuole. Electron-dense, membrane-bound granules were abundant in the proximal segment of normal ductules. After cannulation these granules were still present. It was concluded that the electron-dense granules were insensitive to rete testis fluid and that they did not arise from the fluid leaving the rete testis.

[Determination of substances inhibiting and stimulating the binding of FSH to its receptors]. / Dosage des substances inhibant et stimulant la fixation de la FSH à ses récepteurs.

We describe a method to estimate binding of human 125I-FSH to a preparation of bovine testicular receptors. Various experimental conditions are tested and the validity of the method is demonstrated. Using this method, the presence of biological substances modifying the FSH binding is measured in various fractions of ram retetestis fluid submitted to several steps of purification by chromatography. FSH receptor binding inhibitor (FSHRBI) activity is obtained in a low molecular weight fraction and FSH receptor binding stimulator activity in a larger one. These cybernin activities are isolated in fractions different from the ones observed with inhibin and GnRH like activities.

Peristaltic transport of a physiological fluid. Part - III. applications.

The models of peristaltic flow in non-uniform and uniform tube and channel, developed in the companion papers, Part I and Part II, are applied and compared with the observed flow rates in vas deferens of rhesus monkeys, the small intestine, and the ductus deferens of the male reproductive tract.