Reinitiation of spermatogenesis by exogenous gonadotropins in a seasonal breeder,the woodchuck (Marmota monax), during gonadal inactivity

The present study was undertaken (1) to document structural and functional changes in the testes of seasonally breeding woodchuck during active and inactive states of spermatogenesis and (2) to evaluate the ability of exogenous gonadotropins to reinitiate spermatogenesis outside the breeding season. During seasonal gonadal inactivity, there were significant (P < 0.05) reductions in volumes of several testicular features (testis, seminiferous tubules, tubular lumen, interstitial tissue, individual Leydig cells, Leydig cell nuclei, and Leydig cell cytoplasm) as compared with gonadally active animals. The diameter of the seminiferous tubules was decreased by 26%, and Leydig cell numbers also declined in the regressed testes. These changes were accompanied by a decline in testosterone (T) levels in both plasma and testis, and reduction in epithelial height of accessory reproductive organs. A hormonal regimen was developed that would reinitiate spermatogenesis in captive, sexually quiescent woodchucks. A combination of PMSG and hCG markedly stimulated testicular growth and function and restored spermatogenesis qualitatively. Quantitatively normal spermatogenesis was restored in 2 of 6 treated males. Morphometric analyses revealed substantial increases in seminiferous tubular diameter and in the volume of seminiferous tubules, tubular lumen, total Levdig cells, and individual Leydig cells in the hormone-treated animals. These increased values corresponded to 99, 75, 68, 51, and 200%, respectively, of the values measured in naturally active woodchucks. Leydig cell numbers, however, remained unchanged and approximated only 31% of the number found in naturally active testes. Hormonal stimulation also resulted in a significant rise in serum T as well as in the total content of testicular T, and a marked increase in epithelial height in various accessory reproductive glands. The most effective hormonal protocol for stimulating spermatogenesis was treatment with 12.5 IU of PMSG twice a week for 4 weeks followed by 12.5 IU of PMSG + 25 IU of hCG twice a week for 4 weeks.     

The testis in adreno-leukodystrophy.

Testicular tissue from 7 juvenile and 6 adult patients with adreno-leukodystrophy (ALD) or its adrenomyeloneuropathic (AMN) variant was examined by light and electron microscopy. The seminiferous tubules of the men exhibited hypocellularity, maturation arrest, or Sertoli cells and spermatogonia only. Ultrastructural examination of these specimens revealed vacuolation of Sertoli cell endoplasmic reticulum. Germ cells also demonstrated vacuolation and necrosis, accompanied by slight tubular atrophy and thickening of the tunica propria. Fibrosis or proteinaceous fluid was observed in the interstitium. The tissue of 5 men and 2 boys contained Leydig cells with cytoplasmic striations, which were detectable with the light microscope. A decrease in the number of Leydig cells was noted in some men. All specimens displayed pathognomonic lamellas and lamellar-lipid profiles in mature, immature, or precursor Leydig cells at the ultrastructural level. The Leydig cell demonstrated the primary morphologic defect in the ALD testis; damage to Sertoli cells appeared to be the initial lesion of seminiferous tubules.     

Cysts of the testicular parenchyma and tunica albuginea

Five cases of cysts of the tunica albuginea and two cases of cysts of the testicular parenchyma are described. The cysts of the albuginea were located in the anterior, lateral, and inferior faces of the testis (distant from the epididymis) and covered by a thin albuginea. They extended toward the testicular parenchyma or toward the tunica vaginalis. Their epithelial lining varied from low cuboidal to columnar and consisted of ciliated or nonciliated cells. These cysts were probably of mesothelial origin. The cysts of the testicular parenchyma were also located distant from the mediastinum testis and were not in contact with the albuginea. Their epithelial lining consisted of flattened, cuboidal, nonciliated cells. The origin of the cysts of the testicular parenchyma is not clear. In one case the occurrence of spermatozoa in the cyst lumen and connections with seminiferous tubules suggests an origin in the rete testis.     

Structural characterization and rapid manual isolation of a reptilian testicular tunic rich in Leydig cells.

During the annual breeding season, the testes of the lizard Cnemidophorus gularis are yellow-orange, oviod organs measuring almost 1 cm in greatest diameter. The pigment is confined to the testicular tunic, which contains a zone of Leydig cells and vascular channels more than 50 mu thick. Leydig cells constitute approximately 60% of the zone, with remaining space occupied by capillaries, sinusoids, and lymphatic vessels. Lymphatics are concentrated at the interface between tunic and seminiferous tubules. Interstitial space is poorly developed among the tubules, accounting for less than 3% of tissue volume. Capillaries, lymphatics, and few widely scattering Leydig cells occur in the sparse interstitial space. Leydig cells in the tunic and elsewhere in the testis show ultrastructural features commonly found in mammalian Leydig cells. Separation of the tunic from the seminiferous tubules is achieved in a few seconds by manual decapsulation of the testis and yields an enriched preparation of Leydig cells that is essentially uncontaminated by tubular elements.     

Experimental allergic orchitis in mice

Summary Experimental allergic orchids was induced in (C57BL/6J × A/J)F₁ mice by two injections of syngeneic testicular homogenate emulsified with adjuvants immediately followed by intravenous injection of pertussis vaccine, at a 2 week interval.Histologically, in the initial stage there was occasional focal degeneration and desquamation of both spermatogonia and Sertoli cells within limited parts of the seminiferous tubules, in the peripheral region of the testis. No inflammatory change was present. In some cases, however, inflammatory reaction in the rete testis and focal lymphocytic infiltration in the interstitium were also observed. Subsequently, marked infiltration of lymphocytes, monocytes, and polymorphs were found not only in the testes, but also in rete testis and epididymis. In later stages the inflammatory reaction gradually subsided, but the testes became atrophic due to progression of spermatogenic arrest. Many tubules were lined only with monolayers of Sertoli cells, surrounded by hyperplastic Leydig cells in the interstitium. At 5 months after the 2nd immunization, there was still variable depression of spermatogenesis and hyperplasia of Leydig cells with scattered fibrous scars in the seminiferous tubules, although good regeneration of germ cells appeared in some tubules.Immunological studies revealed that lymphocytes obtained from mice bearing developed orchitis showed a significantly enhanced response in the mixed culture with syngeneic testicular cells, and suggest that cellular immunity plays an important role in the induction of experimental allergic orchitis in mice.     

Structural characterization and rapid manual isolation of a reptilian testicular tunic rich in Leydig cells

During the annual breeding season, the testes of the lizard Cnemidophorus gularis are yellow-orange, ovoid organs measuring almost 1 cm in greatest diameter. The pigment is confined to the testicular tunic, which contains a zone of Leydig cells and vascular channels more than 50 m? thick. Leydig cells constitute approximately 60% of the zone, with remaining space occupied by capillaries, sinusoids, and lymphatic vessels. Lymphatics are concentrated at the interface between tunic and seminiferous tubules. Interstitial space is poorly developed among the tubules, accounting for less than 3% of tissue volume. Capillaries, lymphatics, and a few widely scattered Leydig cells occur in the sparse interstitial space. Leydig cells in the tunic and elsewhere in the testis show ultrastructural features commonly found in mammalian Leydig cells. Separation of the tunic from the seminiferous tubules is achieved in a few seconds by manual decapsulation of the testis and yields an enriched preparation of Leydig cells that is essentially uncontaminated by tubular elements.     

Testicular development in cynomolgus monkeys

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.     

Fate of bone marrow stem cells transplanted into the testis: potential implication for men with testicular failure

To assess adult stem cell differentiation in the testis, we injected bone marrow cells from adult green fluorescent protein (GFP) transgenic mice into the seminiferous tubules and the testicular interstitium of busulfan-treated wild-type or c-kit mutant (W/W(v)) mice. Ten to 12 weeks after transplantation, we examined the fate of the transplanted bone marrow cells and found that they survived in recipient testes. In both the busulfan-treated and W/W(v) mice, some of the GFP-positive donor cells had a Sertoli cell appearance and expressed follicle-stimulating hormone receptor within the seminiferous tubules. In addition, GFP-positive donor cells were found in the interstitium of recipient testes, and they expressed the cytochrome P450 side chain cleavage enzyme (P450scc). In the seminiferous tubules of busulfan-treated mice, GFP-positive donor cells had the appearance of spermatogonia or spermatocytes and expressed VASA. However, this was not found in the seminiferous tubules of W/W(v) mice. We conclude that adult bone marrow cells, in a favorable testicular environment, differentiate into somatic and germ cell lineages. The resident neighboring cells in the recipient testis may control site-appropriate stem cell differentiation. This clinically relevant finding raises the possibility for treatment of male infertility and testosterone deficiency through the therapeutic use of stem cells.     

Cell-cell interactions in the control of spermatogenesis as studied using leydig cell destruction and testosterone replacement

This review centers around studies which have used ethane dimethane sulphonate (EDS) selectively to destroy all of the Leydig cells in the adult rat testis. With additional manipulations such as testosterone replacement and/or experimental induction of severe seminiferous tubule damage in EDS-injected rats, the following questions have been addressed: (1) What are the roles and relative importance of testosterone and other non-androgenic Leydig cell products in normal spermatogenesis and testicular function in general? (2) What are the factors controlling Leydig cell proliferation and maturation? (3) Is it the Leydig cells or the seminiferous tubules (or both) which control the testicular vasculature? The findings emphasize that in the normal adult rat testis there is a complex interaction between the Leydig cells, the Sertoli (and/or peritubular) cells, the germ cells, and the vasculature, and that testosterone, but not other Leydig cell products, plays a central role in many of these interactions. The Leydig cells drive spermatogenesis via the secretion of testosterone which acts on the Sertoli and/or peritubular cells to create an environment which enables normal progression of germ cells through stage VII of the spermatogenic cycle. In addition, testosterone is involved in the control of the vasculature, and hence the formation of testicular interstitial fluid, presumably again via effects on the Sertoli and/or peritubular cells. When Leydig cells regenerate and mature after their destruction by EDS, it can be shown that both the rate and the location of regenerating Leydig cells is determined by an interplay between endocrine (LH and perhaps FSH) and paracrine factors; the latter emanate from the seminiferous tubules and are determined by the germ cell complement. Taken together with other data on the paracrine control of Leydig cell testosterone secretion by the seminiferous tubules, these findings demonstrate that the functions of all of the cell types in the testis are interwoven in a highly organized manner. This has considerable implications with regard to the concentration of research effort on in vitro studies of the testis, and is discussed together with the need for a multidisciplinary approach if the complex control of spermatogenesis is ever to be properly understood.     

Focal orchitis in undescended testes: discussion of pathogenetic mechanisms of tubular atrophy.

OBJECTIVE: To evaluate seminiferous epithelium lesions in adult cryptorchid testes showing lymphoid infiltrates in seminiferous tubules and interstitium (i.e., focal orchitis). Also, to consider the possible role of this lesion in the etiology of tubular atrophy. METHODS: We performed a histopathologic study of the cryptorchid testes and adjacent epididymides removed from 50 adult men who had not been previously treated for cryptorchidism. The study included morphologic and semiquantitative evaluation of seminiferous tubule pathology (according to germ cell numbers), Sertoli cell morphology, tubular lumen dilation, rete testis pattern (normal, hypoplastic, or cystic), and epididymal pattern (normal or epididymal duct hypoplasia). The study also included immunohistochemical evaluation of immune cell markers. The results were compared with clinical and laboratory findings. RESULTS: Focal lymphoid infiltrates (mainly lymphocytes) in seminiferous tubules and interstitium were found in 22 patients (44%), all of whom had unilateral cryptorchidism. The course of orchitis was asymptomatic, and laboratory data were normal. According to the seminiferous tubule pathology, a variety of histopathologic diagnoses, were made: (1) mixed atrophy consisting of Sertoli cell-only tubules intermingled with tubules showing maturation arrest of spermatogonia (11 testes, 4 of which also showed hyalinized tubules); (2) Sertoli cell-only tubules plus hyalinized tubules (4 testes); (3) Sertoli cell-only tubules (3 testes); (4) intratubular germ cell neoplasia (2 testes, 1 of which also showed hyalinized tubules); (5) complete tubular hyalinization (1 testis); and (6) tubular hyalinization plus some groups of tubules with hypospermatogenesis (all germ cell types were present although in lower numbers, 1 testis). Dysgenetic Sertoli cells, that is, Sertoli cells that had undergone anomalous, incomplete maturation, were observed in all nonhyalinized seminiferous tubules with inflammatory infiltrates. Tubular ectasia was observed in 13 cases. The rete testis was hypoplastic and showed cystic transformation in 18 testes, and the epididymis was hypoplastic in 15 testes. CONCLUSIONS: The causes of these focal inflammatory infiltrates are unknown. It is possible that tubular ectasia and Sertoli cell dysgenesis are involved and that these alterations cause a disruption of the blood-testis barrier and allow antigens to enter the testicular interstitium, giving rise to an autoimmune process.     

Immunohistochemical investigations of the autonomous innervation of the cervine testis

The innervation of the cervine testis was studied in 6 roe deers, 7 red deers and 14 fallow deers. The results for the three species are rather similar. With antisera to neurofilament (NF) and neuron specific enolase (NSE), all small and large nerve fascicles can be demonstrated, but single fibers are incompletely stained. Immunoreactions against protein gene product-9.5 (PGP-9.5) and GAP-43 (growth-associated protein-43) are better suited to depict the complete innervation pattern. Bundles of the superior spermatic and inferior spermatic nerves reach the testis via three access routes as funicular, mesorchial and caudal nerve contributions. We found no morphological evidence that the nerves in the cervine testis are directly involved in regulating Leydig cell function or seminiferous tubular motility. The majority of the testicular nerves are associated with the testicular arteries, but the musculature in the walls of the venous plexus pampiniformis is also innervated. All vascular nerve fibers represent postjunctional sympathetic axons displaying a strong dopamine-beta-hydroxylase (DBH) activity, mostly co-expressed with neuropeptide Y (NPY). The presence of cholinergic fibers in the testis of the deer is only sporadic and probably of no functional importance. In all three species of deer, a small quantity of myelinated nerve fibers is encountered in spermatic cord and tunica albuginea and regarded as afferent. The viscerosensory quality in the testicular intrinsic innervation is very likely mediated by the CGRP (calcitonin gene-related peptide)-positive fibers that run independently from the testicular vessels and end in the connective tissue of spermatic cord and tunica albuginea. The testis of the red deer contains significantly more VIP (vasoactive intestinal polypeptide)-positive axons than that of roe and fallow deer. The nerve density in the interior of the testicular lobules shows no regional differences, but there are age- and season-related changes that correlate with the developmental and functional state of the seminiferous tubules. Small testes with solid and narrow tubules, as seen in the prepuberal phase and during seasonal reproductive quiescence, are better innervated than large testes with expanded and spermatogenetically active seminiferous tubules.     

Congenital Leydig cell hyperplasia

The testes and epididymes collected at autopsy from 21 newborns showed apparent Leydig cell hyperplasia which was studied by light and electron microscopy. Twelve newborns were the sons of diabetic mothers, two had undergone rhesus isoimmunization, two were twins of a non-diabetic mother, three had Beckwitz-Widemann's syndrome, and two had leprechaunism. In the first two groups the placentas were also collected and studied. All the testes showed normal seminiferous tubules and diffuse Leydig cell hyperplasia in the testicular interstitium. In addition one son of a diabetic mother and another with Beckwitz-Widemann's syndrome presented multiple Leydig cell nodules in the mediastinum testis and epididymis. The number of Leydig cells per unit area of the testis was calculated on histological sections stained with the peroxidase-anti-peroxidase method for the detection of testosterone. These numbers varied from 1.4 to 3.2 times those found in age-matched controls, except for the two testes with nodular hyperplasia in which the increase in Leydig cells was even greater. The differential diagnosis between Leydig cell hyperplasia, ectopic adrenal cells and leydig cell tumour is discussed. It is proposed that the cause of congenital Leydig cell hyperplasia might be related to placental secretion of human chorionic gonadotrophin.     

Androgen insensitivity syndrome: an immunohistochemical, ultrastructural, and morphometric study

OBJECTIVE: To evaluate the morphometric, immunohistochemical, and ultrastructural lesions of the testes in prepubertal and adult patients with androgen insensitivity syndrome. METHODS: We examined the testicular biopsy using immunohistochemistry for vimentin, smooth muscle actin, and collagen IV antigens. Quantification of seminiferous tubules and testicular interstitium was performed in prepubertal and adult patients with androgen insensitivity syndrome and results were compared with normal testes from both infants and adults. RESULTS: The adult testes presented nodular and diffuse lesions that consisted of Sertoli-cell-only seminiferous tubules. Two types of Sertoli cells could be distinguished, namely, immature vimentin-positive Sertoli cells and nearly mature Sertoli cells. In the nodules, the lamina propria was thin and contained a scant number of actin-positive peritubular cells. Leydig cells were hyperplastic. The prepubertal patients showed only diffuse lesions characterized by Sertoli cell hyperplasia, decreased germ cell numbers, and a discontinuous immunoreaction to collagen IV. CONCLUSIONS: The testicular lesions in androgen insensitivity syndrome are probably caused by primary alterations that begin during gestation. These lesions become progressively more pronounced at puberty, when the nodular lesion pattern (adenomas) is completely developed.     

Reproduction in male swamp wallabies (Wallabia bicolor): puberty and the effects of season

This study describes pubertal changes in testes and epididymides and seasonal changes in the adult male reproductive organs and plasma androgen concentrations of the swamp wallaby (Wallabia bicolor). Pre-pubescent males had testes with solid seminiferous cords and spermatogenesis only to the stage of gonocytes. Their epididymides had empty lumina along their entire length. The testes of three males undergoing puberty had some lumen formation and mitotic activity. Their epididymides were similar in appearance to those of adult males but were entirely devoid of any cells within the lumen of the duct. Three other pubescent males showed full lumen formation in the testes and spermatogenesis up to the elongating spermatid stage. Their epididymides were similar in appearance to those of adult males but with no spermatozoa in the duct. However, cells of testicular origin were found in the lumen of the duct in all regions suggesting that testicular fluids and immature germ cells shed into the rete testes flow through the seminiferous tubules into the epididymis before the release of mature testicular spermatozoa. The weights of testes and epididymides of adult males showed no change throughout the year but prostate weight and plasma androgen concentrations varied significantly with season, with maximums in spring and summer and minimums in winter. The volume fraction of Leydig cells and seminiferous tubules was significantly lower in winter than in summer; but, despite this, maturing spermatozoa were found in the testes throughout the year. Females in the area conceived year-round, suggesting that seasonal changes in the male reproductive tract did not prevent at least some males from breeding throughout the year.     

Morphometric analysis of testicular Leydig cells in normal adult mice

Stereological analysis was carried out on Leydig cells in perfusion-fixed testes of normal adult mice. In a decapsulated testis, the seminiferous tubules occupy 89.3% and the interstitial tissue makes up 10.7% of the volume of the testis parenchyma. The Leyding cells comprise 3.8% of testicular volume. There are 24.9 million Leydig cells per cm³ (or gm) of tissue. An average Leydig cell has a volume of 1,533 μm³ and a surface area of 1150 μm². The smooth endoplasmic reticulum (SER) is the most prominent organelle in the Leydig cells, and has a membrane surface area of 2,428 cm² per cm³ of fresh testis tissue, which is 8.5 times the surface area of the plasma membrane and constitutes 56.9% of the total membranes in Leydig cells. Mitochondria occupy 10.1% of the Leydig cell volume of 11.4% of cytoplasmic volume. The inner mitochondrial membrane (including tubular or vesicular cristae) provides a surface area of about 2855 μm²/cell and is 2.26 times that of the outer membrane. There are approximately 712 cm² of inner membranes per cm³ tissue. Mouse Leydig cells have numerous lipid droplets, which average 147 per cell and occupy 5.1% of the cell volume.     

Analysis of RhoE expression in the testis,epididymis and ductus deferens,and the effects of its deficiency in mice

Rho proteins are a large family of GTPases involved in the control of actin cytoskeleton dynamics, proliferation and survival. Rnd1, Rnd2 and RhoE/Rnd3 form a subfamily of Rho proteins characterized by being constitutively active. The role of these proteins has been studied during the last years in several systems; however, little is known about their expression and functions in the reproductive organs. In this work we analysed the localization and the effect of RhoE deficiency in the testes using mice lacking RhoE expression (RhoE gt/gt), and our research shows some unexpected and relevant results. First, we have observed that RhoE is only expressed in Leydig cells within the testicular parenchyma and it is absent of seminiferous tubules. In addition, RhoE is expressed in the excurrent ducts of the testis, including the ductuli efferentes, epididymis and ductus deferens. Moreover, the testes of postnatal 15‐day‐old RhoE null mice are smaller, both in absolute values and in relation to the body weight. Furthermore, the dimensions of their seminiferous tubules are also reduced compared with wild‐types. In order to study the role of RhoE in the adult, we analysed heterozygous animals as RhoE null mice die early postnatally. Our results show that the testes of adult RhoE heterozygous mice are also smaller than those of the wild‐types, with a 17% decrease in the ratio testis weight/body weight. In addition, their seminiferous tubules have reduced tubular diameter (12%) and a thinner epithelial wall (33%) that appears disorganized and with a swollen lumen. Finally, and probably as a consequence of those alterations, the sperm concentration of heterozygous animals was found to be lower than in the wild‐types. These results indicate that accurate levels of RhoE in the testes are necessary for a correct development and function of male gonads, and suggest novel and unexpected roles of Rnd GTPases in the reproductive physiology.     

Androgen aromatization in cryptorchid mouse testis

Estrogens play an important role in germ cell development. Therefore, we have studied expression patterns of aromatase that converts testosterone into estrogens in 2 recombinant inbred mouse strains that differ in efficiency of spermatogenesis. In order to show whether germ cells are a target for estrogens, estrogen receptors (ER)alpha and beta were localized as well. Adult male CBA and KE mice were made unilaterally cryptorchid to determine alterations in testicular steroidogenesis and spermatogenesis. Differences between control and cryptorchid testes have been studied with respect to (1) cellular sites of aromatase, the enzyme responsible for estrogen formation, (2) the presence of ERalpha and ERbeta in various types of testicular cells, and (3) steroidogenic activity in the testes. Additionally, unilaterally control testes of cryptorchid mice were compared with bilaterally descended testes. Histological or hormonal differences were not found between control testes of cryptorchid and untreated mice. In cryptorchid testes from both strains, degeneration of germ cells was observed as well as a decrease in size of the seminiferous tubules, whereas the amount of interstitial tissue increased, especially in testes of CBA mice. Using immunohistochemistry, aromatase was localized in Leydig cells and germ cells in both control and cryptorchid testes. Sertoli cells were immunopositive in control testes only. In cryptorchid testes of KE mice, aromatase was strongly expressed in spermatids, that were still present in a few tubules. Other cell types in tubules were negative for aromatase. In both control and cryptorchid testes of both mouse strains, ERalpha were present in Leydig cells only, whereas ERbeta were found in Leydig cells and in germ cells in early stages of maturation. In homogenates of testes of CBA control mice, testosterone levels were 3-fold higher than in those of control KE mice, whereas the difference in estradiol levels between both strains was small. Cryptorchidism resulted in decreased testosterone levels and increased estradiol levels. The results of the present study show functional alterations due to cryptorchidism in both mouse strains. Strong aromatase expression in germ cells in control and cryptorchid testes indicates an additional source of estrogens in the testis besides the interstitial tissue and the relevance of estrogen in spermatogenesis.     

Time-dependent changes in post-mortem testis histopathology in the rat

To clarify the contribution of spontaneous or autolytic post-mortem changes to testis histopathology, the testes of adult rats were examined after animals were left at room temperature for 12, 24, 36, and 48 hours postmortem (n = 2 for all time points except 0 hours postmortem, where n = 3). A progressive decrease in testis weight and seminiferous tubule diameter was observed, as well as detachment of the seminiferous epithelium from the basement membrane. As early as 12 hours postmortem, there was observable clumping and margination of chromatin in Leydig cells, Sertoli cells, spermatogonia, spermatocytes, and step 7-10 spermatids; extensive disintegration of Sertoli cells and residual bodies by 24 hours postmortem; and TUNEL positivity of Leydig cells (by 36 hours postmortem) and step 19 spermatids (at 48 hours postmortem). These findings will aid in ensuring proficient histopathological analysis of testes in toxicity studies.     

Localization of cytochrome P450 aromatase and estrogen receptors alpha and beta in testicular cells--an immunohistochemical study of the bank vole

Age- and light-dark cycle-induced changes in immunoexpression of aromatase and estrogen receptors alpha and beta were studied in testes of a seasonally breeding rodent, the bank vole. Seasonal breeding can be mimicked by exposure to different light cycle regimes. In testes of animals that were exposed to long light cycles of 18 h light and 6 h darkness aromatase was in Leydig cells and seminiferous tubules, mainly in spermatocytes, whereas in animals exposed to short light cycles (6 h light and 18 h darkness), only Leydig cells exhibited positive immunostaining for aromatase. Whatever the age of animals, immunostaining for estrogen receptor alpha was restricted to Leydig cells, whereas estrogen receptor beta immunoreactivity was mainly confined to Sertoli cells of both of immature and adult animals, independently of the regimes of light. Additionally, in testes of animals that were exposed to long light cycles, estrogen receptor beta immunoreactivity was observed in seminiferous tubules. Nuclei of germ cells, predominantly spermatocytes and elongated spermatids, were strongly positive which correlated well with aromatase immunoreactivity. As far as we know, the present study is the first study that describes immunoexpression of aromatase and both estrogen receptors alpha and beta in testis of the bank vole. We provide strong evidence that estrogens are not only produced in Leydig cells but also in germ cells in this rodent. These female hormones may play a physiological role in testis, likely in the development of germ cells during spermatogenesis.