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.

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.

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.

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.

5alpha-Reductase and 3alpha-hydroxysteroid oxidoreductase enzyme activities in epididymis and their control by androgen and the rete testis fluid.

The 5alpha-reductase and 3alpha-hydroxysteroid oxidoreductase enzyme activities have been measured in epididymal tissues and the control of these activities by androgens and the rete testis fluid appreciated. The highest 5alpha-reductase enzyme activity was found in the caput, the lowest in the corpus epididymidis. Androgens have a positive control on the 5alpha-reductase but no effect on the 3alpha-hydroxysteroid oxidoreductase activity. Ligation of the efferent ducts decreased significantly both enzyme activities in the caput but not in the corpus or in the cauda epididymidis.

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.

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.

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.

Light microscopic and ultrastructural evidence of epithelial phagocytosis of sperm in the rete testis and ductuli efferentes in the bull.

Light microscopic and ultrastructural observations were made in the bull rete testis and the ductuli efferentes with emphasis on the presence of sperm in the epithelium. Phagocytosed sperm in various stages of degeneration were found in the epithelial cells lining the rete testis and in the nonciliated cells of the ductuli efferentes. Phagocytosis was more prevalent in the rete testis than in the ductuli efferentes. Besides the epithelial cells, degenerating sperm components and residual bodies were in the luminal macrophages of the rete testis. The degeneration of sperm heads presumably progressed in the following order: (i) disruption of the cell membrane, (ii) aggregation of small vesicles, probably of Golgi origin, between the disrupted cell membrane and an outer acrosomal membrane, (iii) loss of the acrosomal matrix, and finally (iv) disintegration of the nuclear chromatin. These degenerative changes probably resulted from increased lysosomal activity of phagocytosing cells. The possible importance and causes of spermiophagy are discussed.

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.

[Effects on the mouse epididymis of juvenile ligation of the ductuli efferentes or proximal epididymal duct: qualitative and quantitative histological studies (author's transl)].

The ductuli efferentes or the epididymal duct between the head and body were ligated in young mice aged 20-30 days, and effects of the ligation on the epididymis were histologically examined by means of qualitative and quantitative procedures at the age of 50-60 days. As reported previously (Takano, 1980), the epididymal duct of the mouse can be divided into five regions (regions I-V). On the morphometric basis, the five regions can be divided into two major portions, proximal and distal. The proximal portion consists of regions I, II and III which form the head of the epididymis. The distal portion is composed of regions IV and V which form the body and tail of the organ. The results of the qualitative and quantitative analysis in the previous study suggested that the proximal portion is functionally related with maturation of sperms and the distal portion is concerned with storage of sperms. Juvenile ligation of the ductuli efferentes or the epididymal duct between the proximal and distal portions caused the following changes in the epididymal duct. 1. Region I, "initial segment", was normally characterized by the lining of high columnar epithelial cells. Juvenile ligation of the ductuli efferentes impaired differentiation of region I, but not that of the remaining regions. Epididymal duct ligation between regions III and IV, on the other hand, exerted no influence on differentiation of the entire duct. Thus differentiation of region I is dependent upon the luminal contents transferred from the testis. 2. The luminal contents were stored in abundance in the distal portion after ligation of the ductuli efferentes, as were in the case of the normal control. On the other hand, the luminal contents were abundant in th proximal portion and scanty in the distal portion after epididymal duct ligation between the proximal and distal portions. These results suggest that the luminal contents may be produced in the proximal portion even after ligation of the ductuli efferentes, transported into the distal portion and stored there. 3. In region IV, the epithelium showed marked histological changes after ligation of the ductuli efferentes or the epididymal duct between regions III and IV. After ligation of the ductuli efferentes, PAS-positive granules and masses frequently appeared in the epithelial cells. After epididymal duct ligation between regions III and IV, clear vacuolated cells and large vacuole-like spaces frequently appeared within the epithelium. Thus it is likely that the epithelial cells of region IV are functionally related to the luminal contents stored.

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.

Sertoli cells secrete both testis-specific and serum proteins.

The secretions of the Sertoli cell were examined with two polyvalent antisera--one prepared against proteins in rat serum and the other against testis-specific proteins in rete testis fluid. These antisera detected 12 serum and 9 testis-specific proteins in rete testis fluid. To determine the origin of these proteins, primary cultures enriched in Sertoli cells were incubated with [35S]methionine, and the radiolabeled proteins in the medium were immunoprecipitated. Gel electrophoresis of the two immunoprecipitates resolved eight serum and nine testis-specific proteins. These two sets of proteins were specifically bound to their respective antiserum and were immunologically distinct. Medium from Sertoli cell cultures contained 10 times more of the testis-specific proteins than did cultures enriched for testicular myoid or interstitial cells. The concentration of the serum proteins in Sertoli cell medium was 5 and 10 times greater, respectively, than in myoid or interstitial cell preparations. The proteins from Sertoli cells were next characterized on two-dimensional gels. Seven of the proteins recognized by antiserum against serum proteins had identical molecular weights and isoelectric points as serum proteins. Three of these proteins were ceruloplasmin, transferrin, and glycoprotein 2. In addition to the proteins immunoprecipitated by the two antisera, more than 60 other proteins were detected on two-dimensional gels of the total secretory proteins. We conclude that the Sertoli cell secretes many proteins, some of which are specific to the testis and others of which are similar to serum proteins.

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.

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.

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.

[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.

Ultrastructural evidence for phagocytosis of spermatozoa in the bovine rete testis and testicular straight tubules.

Ultrastructural examination showed that the epithelium of the bovine rete testis and the tubuli recti could phagocytose spermatozoa. Macrophages were regularly found in the basal parts of the epithelial cells and could be involved in the removal of degraded sperm material.

[Transformation of the rete ovarii into a rete testis and the epoophoron into an epididymis after experimental sex reversal in Gallus domesticus]. / Umformung des Rete ovarii zum Rete testis und des Epoophoron zum Nebenhoden nach experimenteller Geschlechtsumkehr bei Gallus domesticus.

Transformation of the rete ovarii into a rete testis and of the epoophoron into an epididymis after experimental sex reversal in Gallus domesticus is described. After castration of female chicks, the medulla of the left ovary and/or the right gonadal rudiment develop into a testoid or an ovotestis. From general view, this is observed as sex reversal. In case of formation of a testoid, the epoophoron develops into an epididymis and the rete ovarii develops into a rete testis which consists of intra- and extratesticular and intracapsular parts. Thus, a luminated duct system is developed which allows the transport of semen. In case of formation of an ovotestis, the discontinuously side-by-side located parts of the rete ovarii and of the epoophoron are maintained.