The mammalian testicular capsule and its muscle elements

This study of the testicular capsule of rat, dog, cat and human has confirmed the presence of three layers, viz., the tunica vaginalis, the tunica albuginea proper and an innermost tunica vasculosa. Smooth muscle cells are present in the tunica albuginea of all four species and are more prominent at the posterior pole of the testis where the capsule merges with the mediastinum testis. In the rat and the dog, a few striated muscle fibers also are present. While the tunica albuginea is to be considered as a dense connective tissue, the arrangement of the collagen bundles and the presence of a relatively high content of elastic fibers probably permits changes in size of the testis following spontaneous contractions of the muscle elements, which are known to occur. The role of the testicular capsule in sperm transport is discussed in relation to other factors, the spontaneous contractions of the capsule presumably having a “pumping” action and aiding the movement of non-motile spermatozoa from the testis to the epididymis. The presence of striated muscle fibers in two species is of interest and, while these may function in a similar manner to the smooth muscle, they may represent simply an unusual differentiation of embryonic myoblasts.     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).     

Effect of temperature and the role of testicular descent on post-natal testicular androgen production in the mouse.

Testes from mice aged 3, 15, 25, 30 or 60 days were incubated under basal conditions or in the presence of hCG. One testis from each animal was incubated at 37 degrees C while the contralateral testis was incubated at 32 or 34 degrees C. During development total androgen production in response to hCG (at 32 degrees C) showed a marked increase between 15 and 30 days. The major androgens secreted at this time were testosterone and 5 alpha-androstane-3 alpha,17 beta-diol. There was little change in total androgen production between 30 and 60 days but by 60 days testosterone was the dominant androgen. Both basal and hCG-stimulated androgen production were temperature sensitive. These effects were most pronounced at 30 and 60 days with androgen production significantly inhibited at 37 degrees C. To examine the role of testicular descent in regulating steroidogenesis animals were rendered unilaterally cryptorchid at 19 days of age. At 25 days, when descent is normally completed in the mouse, there was no significant difference in steroidogenesis between scrotal and abdominal testes. By 30 days, however, the steroidogenic potential of the abdominal testis was significantly lower than that of the scrotal testis. These results show that testicular steroidogenesis is sensitive to temperature changes around the time of testicular descent, although descent itself is not required to achieve an adult level of steroidogenesis. The results also show, however, that testicular descent is required to maintain the adult level of steroidogenesis.     

Studies on the contractile activity and ultrastructure of the boar testicular capsule

The ultrastructure and the spontaneous and drug-induced contractility of the testicular capsule of 18 boars were investigated. Isometric recordings were obtained in vitro using strips of the tunica albuginea isolated from various regions of the testis. Maximal contractile activity was found in the strips of the posterior border of the testis, in which the histological studies (light and electron microscopy) showed abundant typical smooth muscle cells distributed in layers parallel to the testicular long axis. These cells were largely aggregated in the inner layer of the testicular capsule, which displayed contractile activity similar to that of the entire tunica albuginea. The outer layer of the tunica albuginea was almost totally devoid of smooth muscle fibres and showed little or no contractility. The spontaneous contractions were rhythmic and exhibited an amplitude of 20--70 mg and a frequency of 5--30 contractions/10 min. Norepinephrine, acetylcholine and oxytocin all produced an increase of the contractility of the tunica albuginea, consisting mainly in a rise of the tone.     

The fate of fetal Leydig cells during the development of the fetal and postnatal rat testis

The ultrastructure and developmental fate of the fetal generation of Leydig cells of the rat testis was studied from the 17th day of fetal life up to 100 days after birth. The number of fetal Leydig cells per testis was determined by light microscopic morphometric analysis of semithin plastic sections. In fetal testes (days 17-22 postconception), Leydig cells exhibited a characteristic ultrastructure, containing smooth endoplasmic reticulum, many lipid inclusions and glycogen. Testes of 17-day-old fetuses contained about 25 x 10(3) fetal Leydig cells, rapidly increasing to 90 x 10(3) per testis in 21-day-old fetuses. After birth, fetal Leydig cells per testis remained relatively constant up to 2 weeks (80-90 x 10(3) per testis) and were identified by light and electron microscopy which showed their numerous lipid inclusions, their tendency for clustering and their association with interstitial tissue fibroblasts which partly encapsulated the fetal Leydig cells. From 21-100 days after birth, fetal Leydig cell numbers were quite variable with a mean of 45-60 x 10(3) per testis. These results are the first to show that the fetal generation of Leydig cells persist in the adult testis and do not undergo early postnatal degeneration or dedifferentiation into other interstitial cells. The simultaneous occurrence of the fetal Leydig cells and the adult population of Leydig cells indicates that these cells are distinct cell generations which are developmentally unrelated.     

Dye permeability and alkaline phosphatase activity of testicular capillaries in the postnatal rat

Summary Staining of testicular and epididymal tissues after intravenous, intraperitoneal or subcutaneous administration of a number of dyes was investigated in rats at different stages of postnatal development. After light green injections heavy staining of both testis and epididymis was visible to the naked eye in neonatal animals up to the age of 10 days, while in rats over 15 days old no appreciable staining of the testis could be seen, although the caput epididymis was strongly coloured. From 3–8 hours after subcutaneous acriflavine administration, the nuclei in the blood vessel walls of the testis, as well as the nuclei in the rete testis, tubuli efferentes and caput epididymis, fluoresced in all age groups. The nuclei of the interstitial and tubular cells were stained intensely until the age of 5 days. Thereafter the intensity gradually diminished until the age of 20 days, when no nuclear fluorescence was visible in the seminiferous tubules and even the interstitial nuclei fluoresced weakly or not at all.The histochemical alkaline phosphatase activity of the testicular capillaries was studied by Gomori's method, using fresh and postfixed cryostat sections from postnatal rat testes. The testicular capillaries exhibited appreciable activity at the age of 10 days.On the basis of the present and previous observations on the permeability of the testicular capillaries, the existence of a blood-testis barrier in the puberal and adult rat testis is suggested.Development of the blood-testis barrier and the alkaline phosphatase activity of the testicular capillaries are suggested to reflect general vascular maturation at the beginning of puberty in the rat.Supported by grants from Yrjö Jahnsson's Foundation and P. O. Klingendahl Foundation.     

Identification and characterization of Myosin from rat testicular peritubular myoid cells

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle-alpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4 degrees C, but did at 20 degrees C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1.     

Morphological and immunohistochemical study of testicular capsule and peritubular tissue of emu (<Emphasis Type="Italic">Dromaius novaehollandiae)</Emphasis> and ostrich (<Emphasis Type="Italic">Struthio camelus</Emphasis>)

The testicular capsule and peritubular boundary tissue of the emu and ostrich, as typical representatives of ratite birds, were studied in sexually mature and active birds. The testicular capsule was much thicker (578.1±73.4 μm for the free surface of the ostrich testis, and 176.2±57.5 μm for the emu) than those of members of the Galloanserae. The cellular composition of both testicular capsule and peritubular tissue was similar generally to that of members of the previously studied Galloanserae and of mammals. The tunica albuginea of the testicular capsule mainly comprised smooth-muscle-like or myoid cells mostly running in one direction and occurring in one main mass. Unlike the Galloanserae, the tunica albuginea contained more collagen fibres than smooth muscle cells, especially in the ostrich. Peritubular tissue was similarly composed of smooth-muscle-like cells distributed in several layers. Actin microfilaments and desmin and vimentin intermediate filaments were variably immunoexpressed in these two tissue types in both birds, with a clear dichotomy in the peritubular tissue. Thus, taken together with studies of some members of the Galloanserae, avian testes clearly contain a morphological mechanism that is represented partly by the smooth muscle cells of the testicular capsule and peritubular tissue for transporting the testicular fluid, which is usually copious in birds, and its cellular content from the testis into the excurrent duct system; this mechanism is similar to that found in mammals. The authors are grateful for a University of Pretoria research grant, which aided this work. Dr Peter Ozegbe of the Department of Veterinary Anatomy, University of Ibadan is a recipient of the University of Pretoria Foreign Post-Doctoral Fellowship.     

Smooth muscle and purinergic contraction of the human, rabbit, rat, and mouse testicular capsule

The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in human tissue. Contractions induced by adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP, noradrenaline (NA), acetylcholine (ACh), and electrical field stimulation (EFS) of autonomic nerves were investigated. Nerve-mediated responses of the rabbit and human tunica albuginea were recorded. The EFS-induced human responses were completely abolished by prazosin. In the rabbit, EFS-induced contractile responses were reduced by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid by 36% and by prazosin by 77%. Both antagonists together almost completely abolished all EFS-induced contractions. The human tunica albuginea was contracted by NA, ATP, and alpha, beta-methylene ATP, but not by ACh. The rabbit and rat tunica albuginea were contracted by NA, ATP, alpha, beta-methylene ATP, and ACh. The mouse tunica albuginea was contracted by ACh, ATP, and alpha, beta-methylene ATP, but relaxed to NA. Immunohistochemical studies showed that P2X1 (also known as P2RX1) and P2X2 (also known as P2RX2) receptors were expressed on the smooth muscle of the rodent testicular capsule, expression being less pronounced in man. The testicular capsule of the rat, mouse, rabbit, and man all contain contractile smooth muscle. ATP, released as a cotransmitter from sympathetic nerves, can stimulate the contraction of rabbit smooth muscle. Human, rat, and mouse testicular smooth muscle demonstrated purinergic responsiveness, probably mediated through the P2X1 and/or P2X2 receptors.     

Androgen action via testicular arteriole smooth muscle cells is important for Leydig cell function, vasomotion and testicular fluid dynamics

Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be important for normal testis function as it regulates interstitial fluid (IF) dynamics which is an important intra-testicular transport medium. Androgens control vasomotion, but how they exert these effects remains unclear. One possibility is by signalling via androgen receptors (AR) expressed in testicular arteriole smooth muscle cells. To investigate this and determine the overall importance of this mechanism in testis function, we generated a blood vessel smooth muscle cell-specific AR knockout mouse (SMARKO). Gross reproductive development was normal in SMARKO mice but testis weight was reduced in adulthood compared to control littermates; this reduction was not due to any changes in germ cell volume or to deficits in testosterone, LH or FSH concentrations and did not cause infertility. However, seminiferous tubule lumen volume was reduced in adult SMARKO males while interstitial volume was increased, perhaps indicating altered fluid dynamics; this was associated with compensated Leydig cell failure. Vasomotion was impaired in adult SMARKO males, though overall testis blood flow was normal and there was an increase in the overall blood vessel volume per testis in adult SMARKOs. In conclusion, these results indicate that ablating arteriole smooth muscle AR does not grossly alter spermatogenesis or affect male fertility but does subtly impair Leydig cell function and testicular fluid exchange, possibly by locally regulating microvascular blood flow within the testis.     

Serotonergic innervation of the rat testis

The presence of 5-hydroxytryptamine (5-HT) was determined by h.p.l.c. in perchloric extracts of each isolated compartment of the adult rat testis. The testicular capsule, interstitial cells and interstitial fluid contained 5-HT, but 5-HT was not detected in the tubular compartment. In a group of adult rats, one testis was unilaterally denervated, and the contralateral testis used as control. The superior spermatic nerve, arising from the renal plexus, was excised and 1 week after surgery 5-HT content was measured in the capsule and interstitial fluid of both testes. Denervation caused a significant fall (34%) in 5-HT content. These results indicate that at least part of the testicular 5-HT derives from a serotonergic innervation of the gonad.     

Developmental regulation of galactoglycerolipid and galactosphingolipid sulphation during mammalian spermatogenesis. Evidence for a substrate-selective inhibitor of testicular sulphotransferase activity in the rat.

The synthesis of sulphatoxygalactosylacylalkylglycerol (SGG) is a differentiation marker of mammalian spermatogenesis. Maximal sulphation is observed in rat testis at about 20 days after birth and rapidly declines to low levels as the testis matures. The present data show that this decline in SGG synthesis is due to the appearance of an inhibitor of galactolipid sulphation. The inhibitor is a soluble testicular factor which is first detected at about 25 days after birth. Testicular homogenate can sulphate exogenous galactosylacylalkylglycerol (GG), galactosylceramide (GC) and lactosylceramide (LC) in vitro. The testicular inhibitor is most effective in preventing GG sulphation and inhibits GC and LC sulphation to a lesser extent; this correlates with the finding that glycolipid sulphation shifts from SGG production in 20-day-old testis to GC and LC sulphation at later stages of testicular development. The effect of the inhibitor on sulphotransferase activity from brain and kidney was also determined. The inhibitor decreased the sulphation of GG in vitro by both testis and kidney, inhibited testicular sulphation of GC less effectively and had no effect on GC sulphation by kidney and brain homogenates. A 9500-fold purification of the inhibitory activity has been obtained in a fraction isolated by h.p.l.c.     

Development and differentiation of the rat epididymis. I: ultrastructural aspects of the peritubular zone

We investigated the ultrastructural aspects of the peritubular cells of epididymis and their development from birth to adult age. At birth the peritubular zone consisted of polygonal cells which did not differ from other interstitial cells. Cytoplasmic filaments were visible in the cells of the inner layer at day 6. From day 22 the peritubular cells reached the adult aspect. The peritubular cells in the rat epididymis had aspects similar to those of peritubular smooth muscle cells of rat testis, with a more precocious appearance of cytoplasmic filaments. This finding concurs with the observed precocious contractility of epididymis.     

Use of monoclonal antibody techniques to study the ontogeny of bovine anti-Müllerian hormone

Monoclonal antibodies against bovine anti-Müllerian hormone (AMH) were used to study the hormone in cattle. Anti-Müllerian activity of testicular tissue, immunoreactive testicular AMH, serum AMH concentration and AMH production by incubated testicular tissue were detectable from 42 days, i.e. at the time of seminiferous tubule differentiation, and peaked between 50 and 80 days, when the Müllerian ducts regress in the male fetus. All the values stabilized at a lower level until 30 days after birth and then slowly decreased. At 18 months, only traces of AMH immunoreactivity were detectable in testicular tissue and serum concentration and AMH production by incubated testicular tissue were negligible; the main source of AMH in the adult animal was the rete testis fluid. Study of the disappearance rate of AMH from the serum of castrated calves gave a half-life of approximately 2 days for bovine AMH.     

The Capacity of Testicular Cells of the Postnatal Rat to Reorganize into Histotypic Structures

Cell reorganization experiments in vitro were performed with dissociated rat testes at different ages of postnatal development namely, newborn, 8–10, 18–25, 35–40, and 90 days. Only newborn and juvenile rat testicular cells reassociated into testicular-like organization in rotation culture. Puberal and adult rat testicular cells show morphogenetic organization when they were deprived of germ cells by busulphan pretreatment. A factor present in testicular tissue of puberal and adult rats inhibits reorganization. The inhibitor is confined to the spermatic cell fraction in the testis.     

The cytoskeletal proteins in the contractile tissues of the testis and its excurrent ducts of the passerine bird, Masked Weaver (Ploceus velatus)

The cellular composition of the testicular capsule, seminiferous peritubular tissue, the epithelia as well as periductal muscle cell layers of the excurrent ducts was studied, in sexually mature and active Masked Weaver (Ploceus velatus) birds of the passerine family, Ploceidae. Ultrastructure of the contractile cells in the testicular capsule, peritubular and periductal tissues showed that these cells were smooth muscles of typical morphological characteristics. Variability in the immunohistochemical co-expression of microfilaments and intermediate filaments in the different tissues was evident. Actin and desmin proteins were co-expressed immunohistochemically in the testicular capsule and seminiferous peritubular smooth muscle layer. Actin was singly and very weakly expressed in the rete testis epithelium while cytokeratins and desmin were co-expressed in the epithelium of the excurrent ducts. The periductal muscle layer of all ducts of the epididymis, the ductus deferens as well as the seminal glomus, strongly co-expressed actin and desmin. Vimentin was absent in all cells and tissue types studied. There is clear evidence that the tissues of the male gonad and its excurrent ducts in the Masked Weaver, as has been reported for members of the Galloanserae and Ratitae, contain well-formed contractile tissues whose function would include the transportation of luminal through-flow from the testis into, and through, its excurrent ducts. The microtubule helix in the head and of the mid-piece, of elongating spermatids, as well as of the mature spermatozoa in the various excurrent ducts, including some spermatozoa in the seminal glomus, also co-expressed these three proteins.     

The effect of unilateral castration on plasma and testicular testosterone in rabbits from birth to 60 days.

Male rabbits were hemigonadectomized every 10 days from 1 to 50 days of age and were sacrificed 10 days after the operation. Non-operated controls were sacrificed at the same stages. Plasma and testicular testosterone were quantified by radioimmunoassay. Compensatory testicular hypertrophy was not observed in hemicastrated rabbits. Hemicastration has variable effects on testicular and plasma testosterone levels as a function of age. When it is performed at birth, there is a significant increase in testicular and plasma concentrations of testosterone. Similar results are obtained if the operation is performed at 30 days. Unilateral castration performed at 10, 20 and 50 days reduces testicular and plasma testosterone levels. When hemicastration is performed at 40 days or at the adult age, testicular and plasma testosterone levels are not modified.     

Involvement of testicular DAAM1 expression in zinc protection against cadmium‐induced male rat reproductive toxicity

In order to verify the effects of exposure to Cd and Zn on testicular DAAM1 gene and protein expression and also to ascertain their involvement in the protective role of Zn in prevent the testicular toxicity Cd‐induced in male offspring rats at adult age after gestational and lactational exposure, male offspring rats, from mothers receiving either tap water, Cd, Zn, or Cd + Zn during gestation and lactation periods, were scarified on postnatal days (PND) 70. The reproductive organ (testis, epididymis, and vesicle seminal) were collected, weighed, and analyzed. The results showed that exposure to Cd in utero and through lactation decreased the relative reproductive organ weight, altered the testicular histology at the interstitial and tubular levels, and causing a significant reduction in the daily sperm production (DSP) per testis and per gram of testis, and other then altering the epididymal sperm quality. Furthermore, both mRNA and protein expression of rat testicular DAAM1 were also inhibited in Cd‐treated group. Zn supply has completely corrected the most of these toxic effects. Our results imply that Zn could prevent Cd‐induced testicular toxicity and sperm quality alteration in adult male rat after gestational and lactational exposure, probably via the restoration of the testicular DAAM1 expression inhibited by Cd.     

Spatiotemporal patterns of expression of neurotrophins and neurotrophin receptors in mice suggest functional roles in testicular and epididymal morphogenesis.

Several reports have established that the action of neurotrophins is not restricted to the nervous system but can affect a broad range of non-neuronal cells. Nerve growth factor (NGF) is present in adult testis and has been suggested as a potential regulator of meiosis in rat seminiferous epithelium. Here we present an extensive immunohistochemical study on neurotrophins and their receptors (p75 and trk) in the developing mouse testis and epididymis, and in fetal human testis. During the early steps of testicular and epididymal organization in the mouse, strong p75 immunoreactivity is detectable in the gonadal ridge in the mesenchyme that is excluded from the evolving testicular cords, and in the mesenchymal cells of the mesonephros. Later in organogenesis, most of the p75-positive interstitial cells of the testis coexpress neurotrophin-3 (NT-3) and the truncated trk B receptor in a developmentally regulated pattern. Our Western blot data confirm the expression of these molecules. These findings suggest that neurotrophin receptors play a role in early inductive events during critical periods of testicular and epididymal development. During fetal and postnatal histogenesis, an increasing number of NT-3- and p75-positive mesenchymal cells start to express alpha-smooth muscle isoactin, suggesting a role for the so-called neurotrophic system in the differentiation of testicular myoid cells and epididymal smooth muscle cells. In the testis of an 18-wk gestational-age human fetus, immunohistochemical analysis has shown intense immunoreactivity of mesenchymal cells to antibodies for neurotrophin receptors p75, trk A, and trk C, and their ligands NGF and NT-3. In addition, we found that in the human fetal testis, the interstitial cells that are differentiating into peritubular myoid cells are associated with a dense network of nerve fibers. Our data suggest that neurotrophins and their receptors are involved in a multifunctional system that regulates cell differentiation and innervation in the developing testis and epididymis.