Ultrastructural study of the luminal surface of the ducts of the epididymis of gallinaceous birds

The various ducts of the epididymides of four gallinaceous birds, the turkey (Meleagris gallopavo), domestic fowl (Gallus gallus), guinea-fowl (Numida meleagris) and Japanese quail (Coturnix coturnix japonica) were studied at the scanning and transmission electron microscopy levels. The tissues were fixed either by immersion or vascular perfusion, for comparative purposes. Each duct system, save for a few details, presented similar morphological features in all species. The epithelial surface of the rete testis was regular and each cell bore a single cilium, as well as numerous, or in some parts, very few, short, regular microvilli. Each of the Types I and II non-ciliated cells of the proximal and efferent ducts displayed abundant, moderately long and regular microvilli, and a solitary cilium. The ciliated cells exhibited tufts of cilia. The Type III non-ciliated cell of the connecting and epididymal ducts exhibited a solitary cilium, and numerous microvilli which were intermediate in length between those of the rete testis and those of the efferent ducts. Vascular perfusion of the avian epididymal tissue was the superior method of fixation because it minimised the developments of fixation artefacts. Apocrine secretion did not appear to occur in the epididymis of these birds as the apical blebs of Types I, II and III cells, which have previously been reported, only manifest in this study in inadequately fixed tissues, and were therefore viewed as being artefacts. The present findings suggest that the current terminology, as applied to the avian epididymis, be retained.     

Ultrastructure of parathyroid glands of growing chickens with vitamin D deficiency rickets

Ultrastructural studies were conducted on the parathyroid glands (PTG) of 2- to 7-week-old nontreated control growing broiler chickens (group A, 6 chickens); the same aged chickens with experimentally induced vitamin D deficiency rickets (18 morbid chickens each from groups B, C, and D); and in 4- to 9-week-old chickens naturally affected with rickets (group E, 8 chickens). Four types of principal cells were classified in the PTG of clinically normal birds and in PTG of birds with secondary hyperparathyroidism as follows: Type I cell (small cell in the resting phase); type II cell (medium-sized cell with well-developed rough-surfaced endoplasmic reticulum in the synthesizing phase); type III cell (large cell with well-developed Golgi apparatus and cytoplasmic organelles in the packaging and secretory phase); and type IV cell (medium-sized cell with poor cytoplasmic organelles in the involuting phase). Many coated vesicles were observed in type II and type III cells. In young control chickens, principal cells of the PTG were considered in the resting phase; in older control chickens, there were increased numbers of cells in the synthesizing phase. In the vitamin D deficiency groups, principal cells were in the synthesizing and packaging and secretory phases. In birds with naturally occurring rickets, principal cells were in packaging and secretory phases, with a small number of cells in the involuting phase. The ultrastructural changes of the PTG from the initial to final phases in growing chickens with secondary hyperparathyroidism were demonstrated.     

Immunolocalization of sex steroid receptors in the epididymis and ductus deferens of immature and mature Japanese Quail, Coturnix Japonica

The goal of this study was to determine whether in the Japanese quail the male genital tract contains receptors for progesterone, androgen and estrogen (PR, AR and ER, respectively), which have significant roles in reproductive functions, and whether their localization changes during sexual maturation. The epididymis and ductus deferens (middle and ampulla regions) of immature (approximately 30-day-old) and mature male Japanese quail were collected and frozen sections of them were immunostained for PR, AR and ER. The immunoreaction products for AR and PR were found in the nuclei of epithelial cells in the efferent ductules, epididymal duct, and the middle and ampulla regions of the ductus deferens of mature and immature birds. In the mature birds, the epithelial cells of the efferent ductules, epididymal duct, and the middle and ampulla regions of the ductus deferens were positive for ER, although some of the cells in the ductus deferens were negative. The epithelial cells of the ductules in the epididymis stained positive for ER, but the immunoreactions were negligible in the ductus deferens of immature birds. These results suggest that the epididymis and ductus deferens in quail possesses PR, AR and ER receptors. Each receptor is expressed before sexual maturation, although enhancement of ER expression may occur during maturation.     

Structural and immunohistochemical features of the epididymal duct unit of the ostrich (Struthio camelus)

The epididymal duct unit, comprising the ductus conjugens, ductus epididymidis and ductus deferens, was studied histologically, ultrastructurally and immunohistochemically in five sexually mature and active birds. The main morphological features of the pre-dominant non-ciliated (type III) cell of the epithelial lining of this duct unit include, but are not limited to, a moderately abundant smooth or sparsely granulated endoplasmic reticulum, electron-dense secretory granules and numerous mitochondria in the supranuclear zone of the cytoplasm. A single, large heterogeneous lipid droplet, of unknown function, was characteristically situated immediately proximal to the nucleus. The epithelium is obviously secretory and specifically, of the merocrine, and not apocrine, type of secretion. The epithelium of the epididymal duct unit was only focally and weakly to moderately immunopositive to both actin MF and desmin IF, while the duct unit was immunonegative to cytokeratin and vimentin intermediate filaments. The peritubular muscular layer was moderately to strongly positive to both actin and desmin, and negative to cytokeratins and vimentin.     

Immunohistochemical study of galectin-3 in mature and immature bull testis and epididymis

Galectin-3, a member of the β-galactoside-binding protein family, has been implicated in mammalian sperm maturation. We examined galectin-3 expression in the testis and epididymis of sexually mature and immature bulls. Western blot analysis showed varying levels of galectin-3 in the bull testis and epididymis, and galectin-3 immunoreactivity was higher in the mature testis and epididymis than in immature organs. Galectin-3 was primarily localized in interstitial cells of the immature bull testis and in the peritubular myoid and interstitial cells of the mature testis. In the immature epididymis head, galectin-3 was primarily in the principal and basal cells of the epithelium. In the mature epididymis head, moderate levels of galectin-3 were detected in the sperm, while low levels were found in the stereocilia, epithelium and connective tissue. In the immature epididymis body, moderate protein levels were detected in the principal cells, while lower levels were found in the basal cells. The mature epididymis body showed moderate levels of galectin-3 immunostaining in the stereocilia and epithelium, but low levels in the connective tissue. In the immature epididymis tail, only low levels of galectin-3 staining were found in the epithelium, whereas the mature epididymis tail showed high levels of galectin-3 in the principal cells, moderate levels in the basal cells and low levels in connective tissue. These findings suggest that galectin-3 expression plays a role in the maturation and activation of sperm in bulls.     

A light and scanning electron microscopic study of the epididymis active state of the endemic Mexican rodent Peromyscus winkelmanni (Carleton) (Rodentia: Muridae)

The macroscopic and microscopic anatomy of the epididymis of the sexually mature Peromyscus winkelmanni (carleton) was examined using light and scanning electron microscopy. The epididymis was divided into three regions: caput, corpus and cauda. The epididymal duct was lined with columnar and cubic epithelium with stereocilia and covered by a muscular connective tissue sheath. Capillaries appear to penetrate directly into the epithelium from the underlying connective tissue in the initial segment. The epididymal epithelium presents four cell types: principal, basal, apical and clear cells. Based on morphological differences (height of epithelial cells, length of the stereocilia, luminal area, larger diameter and spermatic index), the epididymis of P. winkelmanni, presents seven zones. The stereocilia of the epididymal ducts of zones I, II, IV and V are thick and tall, while in zone III they are thin and short. The stereocilia in zone VI are thin, while in zone VII they are short but thick. The secretory products observed in the lumen of the epididymal ducts have vesicular, granular and fibrous form in the seven zones. This study contributes to an understanding of the morphofunctional features of the epididymis in sperm maturation in a species that shows seasonal reproductive activity.     

Expression of chicken LEAP-2 in the reproductive organs and embryos and in response to <Emphasis Type="Italic">Salmonella enterica</Emphasis> infection

In recent years host antimicrobial peptides and proteins have been recognised as key mediators of the innate immune response in many vertebrate species, providing the first line of defense against potential pathogens. In chickens a number of cationic antimicrobial peptides have been recently identified. However, although these peptides have been studied extensively in the avian gastrointestinal tract, little is known about their function in the chicken reproductive organs and embryos. Chicken Liver Expressed Antimicrobial Peptide-2 (cLEAP-2) has been previously reported to function in protecting birds against microbial attack. The aim of this study was to investigate the expression of cLEAP-2 gene in the chicken reproductive organs, as well as in chicken embryos during embryonic development, and to determine whether cLEAP-2 expression in the chicken reproductive organs was constitutive or induced as a response to Salmonella enteritidis infection. RNA was extracted from ovary, oviduct, testis and epididymis of sexually mature healthy and Salmonella infected birds, as well as from chicken embryos until day ten of embryonic development. Expression analysis data revealed that cLEAP-2 was expressed in the chicken ovary, testis and epididymis as well as in embryos during early embryonic development. Quantitative real-time PCR analysis revealed that cLEAP-2 expression was constitutive in the chicken epididymis, but was significantly up regulated in the chicken gonads, following Salmonella infection. In addition, expression of cLEAP-2 during chicken embryogenesis appeared to be developmentally regulated. These data provide evidence to suggest a key role of cLEAP-2 in the protection of the chicken reproductive organs and the developing embryos from Salmonella colonization.     

Effects of age and gonadal steroids on the localization of antigen presenting cells in the epididymis of the male chicken, Gallus domesticus

The goal of this study was to localize antigen presenting cells (APC), which may play roles in defense against pathogens and fertility, and examine the effects of age and gonadal steroids on their population in the rooster epididymis. Healthy White Leghorn male birds (immature 60-day-old birds; matured 150-, 330-, and 550-day-old), and immature birds treated with testosterone propionate (TP) or estradiol benzoate (EB) for 3 or 6 days were used. Cryostat sections of the epididymis and ductus deference were immunostained for Ia to identify APC. RT-PCR was performed to confirm the expression of major histocompatibility complex class II (MHC class II) mRNA in the epididymis. Ia+ cells were localized in the surface epithelium and subepithelial layer of the ductules and occasionally in the luminal content of the epididymis and ductus deference. RT-PCR analysis confirmed expression of MHC class II mRNA in the epididymis, ductus deferens, testis, and spleen. The frequency of Ia+ cells in the subepithelial layer was significantly greater in the proximal efferent ductules than in the other two types of ductules in the epididymis of 550-day-old birds. Although there were no significant differences in the frequencies in the subepithelial layer of the proximal efferent ductules between 60- and 150-day-old birds, the frequencies were significantly greater in 330- and 550-day-old birds than in 60-day-old birds. The frequencies of Ia+ cells in the ductus deferences was increased in the 150-day-old birds compared with the 60-day-old birds, with a larger increase in 330- and 550-day-old birds. The Ia+ cell frequency was significantly increased by EB-injection, but not by TP-injection, on Days 3 and 6 of injection compared with Day 0. These results suggest that the population of APC in the epididymis increases with age after sexual maturation, and estrogen may be one of the factors involved in induction of Ia+ cells.     

Effects of temperature and pH on osmotic fragility of erythrocytes of the domestic fowl (Gallus domesticus) and guinea-fowl (Numida meleagris).

The effects of temperature and pH on the osmotic behaviour of erythrocytes of the domestic fowl and guinea-fowl were studied. Erythrocytes of the guinea-fowl were observed to be more osmotically fragile than those of the domestic fowl at 29 degrees C and pH 7.7. The osmotic fragility of erythrocytes of both species decreased as the temperature (at constant pH of 7.7) or pH (at constant temperature of 29 degrees C) increased. The erythrocyte count, packed cell volume, haemoglobin concentration and mean corpuscular volume were identical in both species.     

Sonographic evaluation of thyroid morphology during the normal estrous cycle in the Indo-Pacific bottlenose dolphin (Tursiops aduncus)

Physiologic changes occurring in the thyroid in response to the estrous cycle have been noted in companion animals. However, in bottlenose dolphins, the influence of different reproductive states on thyroid morphology remains unclear. Sonography was used to evaluate the variations of thyroid morphology for nine consecutive estrous cycles of four sexually mature, female Indo-Pacific bottlenose dolphins (Tursiops aduncus). The estrous cycle was divided into two phases for evaluation: the follicular and luteal phases. To compare changes associated with sex, thyroid volumes were measured in the different phases during the estrous cycle of female dolphins and were compared to the ultrasound examinations of the four male dolphins measured over a 2-mo period. In males, there was no significant difference in thyroid volume during the study period (P > 0.05). The thyroid volume measured in combining all estrous cycle phases of the female dolphins was significantly larger than that measured in the male dolphins (P < 0.05). A difference in thyroid volume during the estrous cycle was observed, with the thyroid volume during the follicular phase significantly smaller (P < 0.05) than that of the luteal phase, and is possibly related to the influence of female sex steroids. Thyroid volume variability during estrus should, therefore, be taken into account when examining the thyroid gland of female dolphins.     

Transcriptional profiling of antimicrobial peptides avian β-defensins in the chicken ovary during sexual maturation and in response to Salmonella enteritidis infection

Avian β-defensins (AvβDs) are antimicrobial peptides that play significant roles in the innate immune system in chickens. The aim of this study was to identify the types of AvβDs expressed in the chicken ovary, to investigate the effects of sexual maturation in the ovarian mRNA abundance and to determine the changes in their expression levels as a result to Salmonella enteritidis (SE) infection. RNA was extracted from the ovary of healthy prepubertal, sexually mature and aged birds, as well as from sexually mature and aged SE infected birds. Real-time PCR analysis revealed that 11 AvβDs genes were expressed in the chicken ovary. A significant up regulation of AvβD1, 3, 4, 5, 7 and 11 was observed in the ovary of sexually mature and aged birds. Furthermore, a significant up-regulation of AvβD4, 5, 7, 11 and 12 was observed in the ovary of SE infected sexually mature birds. These results suggest that the mRNA expression of at least six AvβDs increase with age in the ovary of laying hens, and that at least five AvβDs show an induction in their expression in response to SE infection, indicating an AvβD-mediated immune response mechanism in the chicken ovary.     

Development, validation, and utilization of a novel antibody specific to the type III chicken gonadotropin-releasing hormone receptor

Two gonadotropin-releasing hormone receptors (GnRH-Rs) have been characterized in chickens to date: cGnRH-R-I and cGnRH-R-III, with cGnRH-R-III being the predominant pituitary form. The purpose of the present study was to first validate a novel antibody for the specific detection of cGnRH-R-III and second, using this antibody, detect changes in cGnRH-R-III protein levels in the pituitary gland of male and female chickens during a reproductive cycle. The localization of cGnRH-R-III within the anterior pituitary gland was also determined. Western blotting of pituitary extracts and transiently transfected COS-7 cell lysates revealed that our antibody is highly specific to cGnRH-R-III protein. Similarly, when used in immunocytochemistry, this antibody specifically detects cells expressing cGnRH-R-III and not cGnRH-R-I. Western blot analyses of chicken pituitary gland homogenates show that cGnRH-R-III protein levels are significantly greater in sexually mature birds than in immature birds or birds at the end of a reproductive cycle (P < 0.0001). A similar pattern was observed for both males and females. Additionally, the antibody was able to detect cGnRH-R-III in cells along the periphery of the cephalic and caudal lobes of the anterior pituitary where the cells containing the gonadotropins are located. In summary, we successfully validated a novel antibody to cGnRH-R-III and showed levels of cGnRH-R-III protein in the pituitary fluctuate with respect to the reproductive status in both male and female chickens.     

The surface features of the epithelial lining of the ducts of the epididymis of the ostrich (Struthio camelus)

The luminal appearance of the various ducts of the epididymis of the ostrich was studied by scanning electron microscopy in tissues fixed by immersion in glutaraldehyde. The ductal types were similar to those previously described for some other species of birds. Numerous short microvilli, as well as a single cilium, projected from the apical surface of the rete testis cell. The ciliated cells of the efferent ductules projected tufts of cilia into the ductal lumen, while the non-ciliated cells bore short microvilli. The connecting and epididymal ducts were lined by a columnar cell type whose apical surface bore uniformly distributed microvilli and a single, centrally situated cilium. The spermatozoa found in all ducts of the epididymis bore a distal cytoplasmic droplet. This observation has implications for the maturational process in the ostrich spermatozoon in the epididymis. The surface features of the ducts, except for a few noteworthy differences, were generally similar to those previously described for the male domestic fowl, turkey and duck.     

Fine Structure of Principal Cells in the Initial Segment of the Epididymal Duct in the Ram

The aim of the study was to investigate cytological characteristics of principal cells in the proximal caput epididymidis of the ram. Young, sexually mature rams were used. The epididymis was fixed by local vascular perfusion with glutaraldehyde. Samples for histology were embedded in paraffin wax. Small samples for electron microscopy were immersed in osmium tetroxide and embedded in Epon. Signs of pinocytosis and vesicular merocrine secretion were seen, as well as numerous small basal lysosomes. The distal end of the segment showed lower cells with little evidence of secretion.     

二乙基己烯雌酚对成年仓鼠附睾肥大细胞数量分布的影响

成年雄性仓鼠经皮下连续注射二乙基己烯雌酚(DES)7 d后,用改良甲苯胺蓝染色法(MTB)和阿尔新蓝番红染色法(AB S)研究DES对附睾肥大细胞的形态大小、类型和数量分布的影响。结果表明:仓鼠附睾肥大细胞为结缔组织型肥大细胞,多分布于附睾头与附睾尾的被膜和间质中。试验组的肥大细胞常见于附睾管近旁,多呈脱颗粒状。虽然试验组与对照组的肥大细胞大小相近,但试验组的肥大细胞数量增多,尤其是附睾尾间质中肥大细胞增加明显(P<0.01)。     

达乌尔黄鼠精子形成和细胞色素芳香化酶免疫位置的季节变化

用组织学和免疫组织化学方法调查达乌尔黄鼠精子形成季节性变化和细胞色素芳香化酶(P450 arom)在精巢和附睾中的免疫位置。黄鼠繁殖期与非繁殖期精巢大小、重量、生精小管直径存在显著差异;黄鼠繁殖期精巢中存在从精原细胞到有尾精子各期生殖细胞,非繁殖期精巢中只存在精原细胞和初级精母细胞。另外,繁殖期黄鼠附睾管中存在大量有尾精子,而非繁殖期附睾中未见精子存在。繁殖期P450 arom在黄鼠精巢的间质细胞、支持细胞、精子细胞和附睾头部输出小管上皮细胞都有发现,而在非繁殖期没有发现它的活力。这些结果表明达乌尔黄鼠精子形成、成熟是伴随着精巢复发和退行呈现显著季节性变化,雌激素在精子形成和成熟过程中起着重要的生理性作用。     

Localization of Oestrogen Receptors in the Epididymis During Sexual Maturation of the Domestic Cat

Oestrogens are involved in regulation of spermatogenesis and sperm maturation and are essential for male fertility. To study the role of oestrogens on epididymal function in the domestic cat, we analyzed the localization patterns of oestrogen receptors (ERs) within the epididymis of juvenile, pubertal and adults using immunohistochemistry. Cat epididymal tissues obtained during routine castrations were fixed in chilled Bouin's solution and processed for immunohistochemistry with ER-specific antibodies. For a certain receptor type, ER localization was influenced by donor age. In the juvenile epididymis, ERα was localized in the nuclei of epithelial cells of efferent ducts and undifferentiated epithelium of the ductus epididymis. During puberty, ERα localization in the undifferentiated epithelium of the epididymis shifted from the nuclei to the cytoplasm and plasma membrane. Oestrogen receptor-α level was highest in the pubertal and adult epididymis, especially within the cytoplasm and in plasma membranes of caput epithelial cells. This finding was suggestive of a role in fluid reabsorption within the efferent ducts and the epididymis. In corpus and cauda regions, ERα was less abundant, suggesting a minor role for oestrogens in sperm storage areas. Interestingly, localization of ERβ was neither influenced by age nor location within the epididymis and was ubiquitous throughout. Results demonstrate that oestrogen actions within the epididymis may be predominantly mediated through ERα during sexual maturation in the domestic cat.     

An unusual mechanism of disposal of superfluous spermatozoa in vasectomised quails

As part of a series of studies designed to evaluate usual and potential functions of epithelial cells in the avian epididymis under varying circumstances, adult, sexually active male Japanese quails were vasectomised for 3, 6 and 8 weeks. The testes, epididymides and deferent ducts were studied histologically and ultrastructurally. Testicular lesions were minimal, but epididymal response was profound. Non-ciliated (type I) cells of the proximal efferent duct (PED) actively phagocytized spermatozoa and their fragments. An especially noteworthy and apparently novel observation was the proliferation of the lining epithelium in parts of the PED to form additional adluminal sheets of spermiophagic epithelium which did not lie directly on basal laminae. These sheets were composed exclusively of non-ciliated cells. Spermatozoal dissolution and macrophage activity were also important mechanisms for the disposal of superfluous and incarcerated spermatozoa in the epididymis of this bird.     

Effects of age, sex and housing on the trabecular bone of laying strain domestic fowl.

To determine the effects of age, sex and housing on trabecular bone volume, samples were collected from groups of male and female domestic fowl housed in cages or floor pens from four to 60 weeks old. Between 25 and 60 weeks old, trabecular bone volume decreased by 25 per cent in sections of free thoracic vertebrae (T5) from female birds, the loss occurring at an earlier age in caged birds. Over the sample period, TBV in male caged birds diminished by 35 percent, but male floor birds showed no reduction in trabecular bone volume. At 60 weeks, trabecular bone volume was 30 per cent greater in male caged birds and 40 per cent greater in male floor birds than in the corresponding females. In reproductively active females, no trabecular osteoid was observed, indicating no new trabecular bone formation. However, trabecular osteoid was present in two birds aged 60 weeks which had regressed ovaries. Osteomalacia was not seen in any of the bone samples.     

Expression of lactoferrin in the boar epididymis: effects of reduced estrogen

Lactoferrin is regulated by estrogen in the female reproductive tract and evidence in immature mice suggests that it may be estrogen regulated in males as well. The estrogen regulation of lactoferrin in the epididymis of the boar, a high estrogen-producing male, is unknown. This study was designed to test the hypothesis that lactoferrin expression in the boar epididymis is regulated by estrogen. Twenty-one littermate pairs of boars were treated with vehicle or Letrozole, an aromatase inhibitor, from 1 week of age until castration at 2 through 8 months. Epididymal tissue was collected at castration and fixed for immunolocalization of lactoferrin. Epididymal and testicular tissues were also collected from five mature boars (1-2.5 years) and fixed for immunocytochemistry (ICC). Lactoferrin was localized in the principal cell cytoplasm of the caput, corpus and cauda of developing boars but only in the corpus and cauda of mature boars. Basal cells were negative for lactoferrin. Sperm in the corpus and cauda was also positive for lactoferrin. The efferent ducts and testes were negative for lactoferrin. Intensity of lactoferrin immunostaining increased with age in the corpus and cauda regardless of treatment. Reduced endogenous estrogen in the epididymis during development did not affect the intensity of immunostaining between control and Letrozole-treated animals. Lactoferrin expression in the epididymis of the developing boar does not appear to be regulated by estrogen.