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The localization of immobilin, a glycoprotein known to be present and to immobilize spermatozoa in the lumen of the epididymis, was investigated using light and electron microscope immunocytochemistry. In the light microscope, a distinct immunoperoxidase reaction product was observed in the lumen over the brush border of the epithelial nonciliated cells of the efferent ducts, while only a faint reaction was seen over their supranuclear region. In the proximal area of the initial segment of the epididymis no immunoperoxidase staining was observed either over epithelial cells or in the lumen. In the middle area of the initial segment, several epithelial principal cells became intensely immunostained but the majority were unstained; a weak reaction appeared in the lumen. In the distal area of the initial segment, more principal cells became immunostained, and while some were intensely reactive, others were moderately or weakly stained or unreactive. In the intermediate zone and proximal caput epididymidis, the principal cells showed the maximal immunoreactivity with all principal cells being reactive; staining in the lumen also reached its maximal reactivity in these areas. Immunostaining of principal cells gradually decreased along the epididymal duct and disappeared in the cauda epididymidis, however, an intense reaction persisted in the lumen. In the distal area of the cauda epididymidis, clear cells were reactive. In the electron microscope, immunogold labeling of reactive principal cells of the middle and distal areas of the initial segment, intermediate zone, and caput epididymidis was detected over cisternae of endoplasmic reticulum, stacks of Golgi saccules, and spherical electron lucent (200-400 nm in diameter) vesicles. The latter were present on the trans face of the Golgi stack, in the vicinity of th Golgi apparatus, and close to the apical cell surface; they are considered as secretory vesicles involved in the secretion of immobilin. In the distal area of the cauda epididymidis, epithelial clear cells showed an intense immunogold labeling over their endocytic apparatus. Immunogold labeling in the lumen of the epididymis was found over a fine flocculent material dispersed between the sperm. This material was especially abundant in the cauda epididymidis and did not appear to be bound to the surface of the sperm. The present results suggest that principal cells of the epididymis are involved in the secretion of immobilin, but that a differential secretory pattern exists between epididymal segments with maximal secretory activity occurring in the intermediate zone and proximal caput epididymidis, while no secretion takes place in the cauda epididymidis. Excess immobilin appears to be endocytosed for degradation by clear cells of the cauda epididymidis.
Anat Rec 1992 Feb
PMID:Epithelial cells of the epididymis show regional variations with respect to the secretion of endocytosis of immobilin as revealed by light and electron microscope immunocytochemistry. 154

The distribution of filipin-sterol complexes (FSCs) and intramembranous particles (IMPs) in the plasma membrane of the late spermatid of the boar and of the sperm obtained from the epididymides, ejaculates, and uterus 2 hours after mating was examined by a freeze-fracture replica technique. In the late spermatid, the FSC density was found to be very low. A majority of the FSCs in the acrosomal plasma membrane (APM) appeared as protuberances on the E face in the epididymal, ejaculate, and uterine sperm. The density of the FSCs in the principal segment (PS) of the APM was 291 +/- 44 FSC/microns2 (mean +/- standard deviation, S.D.), 322 +/- 41 FSC/microns2 and 355 +/- 31 FSC/microns2 in the caput, corpus, and cauda epididymidis, respectively. In comparison with the cauda epididymal sperm, the FSC density gradually decreased in the PS of the ejaculated (277 +/- 39 FSC/microns2) and uterine sperm (243 +/- 50 FSC/microns2). The reduction was especially remarkable in the equatorial segment (ES), where the density of FSCs in ejaculated and uterine sperm decreased to about half and less than half of that in the cauda epididymal sperm, respectively. Large (13 nm) and small (8 nm) IMPs were distributed evenly and densely in the P face of the APM in the late spermatid, epididymal, and ejaculated sperm. In the uterine sperm, IMP-free areas were observed in the P face of the plasma membrane, a feature thought to represent one of the capacitation changes of the boar sperm.
Anat Rec 1992 Feb
PMID:Changes in the distribution of filipin-sterol complexes in the boar sperm head plasma membrane during epididymal maturation and in the uterus. 154 1

Actin, alpha-actinin, and tropomyosin were localized in the testicular, epididymal, and ejaculated spermatozoa and in the epithelium of the bovine epididymis by means of specific antibodies using an indirect immunofluorescence technique. Immunocytochemical results were confirmed by the western blot analysis. Independent of the method of fixation, washing, or sonication, actin, alpha-actinin, and tropomyosin were all consistently localized in the neck of the spermatozoa. Actin and tropomyosin present in the postacrosomal area could be removed by sonication, whereas alpha-actinin in the basal plate appeared to be resistant to the treatment. In the unwashed spermatozoa alpha-actinin-specific immunofluorescence was seen over the acrosomal area, whereas in the washed sperm it appeared as a narrow cap at the margin of the head. In the latter location, its distribution was similar to that of tropomyosin. In the majority of preparations, tropomyosin could be localized in the principal piece of the tail. Even though some actin-specific immunofluorescence could be identified in the principal piece of the tail of the testicular and epididymal spermatozoa, a strong immunoreaction appeared only in the ejaculated spermatozoa. In the principal cells of the epididymal epithelium, specific fluorescence for actin, alpha-actinin, and tropomyosin occurred in the apical junctional complex. Basal bodies of the solitary cilia of the epididymal epithelium were labelled with antitropomyosin and anti-alpha-actinin antibodies. Besides offering new information about the cytoskeletal composition of the mammalian sperm, the present results support the hypothesized homology between the connecting piece of the sperm neck and the basal body of the cilia.
Anat Rec 1992 May
PMID:Localization of actin, alpha-actinin, and tropomyosin in bovine spermatozoa and epididymal epithelium. 160 79

The light and electron microscopic appearance of the various epithelial cells lining the efferent ducts and different regions of the epididymis were examined in rats on postnatal days 21, 39, 49, 56, and 90 to determine the role of androgens and/or spermatozoa, as well as other possible factors, on the structural differentiation of these cells. Five conclusions may be drawn from the observations made. First, on day 21 epithelial cells of all regions are structurally undifferentiated. Second, it was not until day 49 that nonciliated cells of the efferent ducts resembled those of adult animals, suggesting that more than one factor, such as androgens, testicular products, and/or spermatozoa, is needed for their full structural differentiation. Third, principal cells of the epididymis become structurally differentiated by day 39, i.e., these cells contained an elaborate Golgi apparatus, endoplasmic reticulum cisternae, and numerous 200-400 nm electron lucent secretory vesicles, as well as a full complement of endocytic organelles; this occurred in spite of the absence of spermatozoa in the epididymal lumen. The differentiation of these epididymal cells may be under the influence of androgens, which are known to be high at this time, but may also be due to specific secretions from Sertoli cells secreted directly into the efferent ducts. Fourth, clear cells of the cauda epididymidis are fully differentiated by day 39. The presence of degenerating germ cells in the lumen of the cauda epididymidis and various cellular debris, as well as high androgen levels, may be factors causing the differentiation of the cells of this region. Finally, clear cells of the corpus and cauda epididymidis only become fully differentiated by day 49, at a time when spermatozoa appear in the lumen, despite high levels of androgens at day 39; this observation indicates that the presence of spermatozoa in the lumen may be a necessary factor in causing their differentiation. Overall, these results suggest that a combination of different factors are necessary for the structural differentiation of the various epithelial cell types of the different regions of the epididymis.
Anat Rec 1992 Jun
PMID:Structural differentiation of the epithelial cells of the testicular excurrent duct system of rats during postnatal development. 160 86

We studied in the rat epididymis the presence of membrane-bounded vesicles in the stereociliar areas of the epithelial cells. The intimate contact between principal cell stereocilia and luminal spermatozoa was also explored. The epididymidis of adult male albino rats were fixed with Mollenhauer's fixative via the thoracic aorta; they were removed and the caput and the cauda were separated and fixed for 4 additional hours at 4 degrees C. After fixation, the samples were processed with routine techniques for transmission and scanning electron microscopy. The study showed membrane-bounded vesicles in the lumen of the caput and cauda epididymidis. They are present between stereocilia, in the most peripheral regions of the epididymal lumen, and in a stereocilia-free zone in the apical plasma membrane of the principal cells. The smaller vesicles are located near the apical surface of the latter, and the larger ones are located near the tips of the stereocilia. Their contents are electron lucent in some images and electron dense in others. In several thin sections some of the vesicles are observed to have a stalk. This suggests that the possible mode of production may be an exocytotic process. Some membrane-bounded vesicles were found to be in contact with the head or the tail of maturating spermatozoa. Moreover, an intimate contact was found to exist in the epididymidis between the plasma membranes of the spermatozoa and the stereocilia. These observations seem to suggest two possible mechanisms for sperm-epididymal cell relations: 1) release of a secretion product via the membrane-bounded vesicles and 2) direct contact between stereocilia and spermatozoa.
Anat Rec 1991 Oct
PMID:Interactions between rat epididymal epithelium and spermatozoa. 174 19

The distribution of actin and CaM in hamster spermatozoa was examined during the early events of fertilization using postembedding immunogold procedures. Actin was immunolocalized with a polyclonal antibody and two monoclonal antibodies. CaM was immunodetected with a polyclonal antibody. In epididymal sperm, actin labeling was found solely in the principal piece of the flagellum. CaM labeling was observed in the postacrosomal lamina, subacrosomal ring, and tip of the perforatorium. These distributions were not modified after capacitation and acrosome reaction. During the successive steps of sperm-egg fusion actin remained undetected in the sperm head whereas its location did not change in the flagellum. CaM distribution remained unmodified until the sperm head begins to decondense. At later stages of sperm head decondensation the postacrosomal lamina and its CaM labeling disappeared, whereas gold particles were still detected in the subacrosomal layer. The predominant location of actin into the egg cortex, particularly the microvillus-free area was confirmed. Except for the CaM labeling of the meiotic spindle, no special CaM location could be found throughout the egg. Thus, in hamster, a role for sperm actin in sperm-egg fusion appears unlikely. In contrast the CaM present in the Ca(2+)-rich postacrosomal lamina could be involved in the regulation of egg activation.
Anat Rec 1991 Nov
PMID:Sperm actin and calmodulin during fertilization in the hamster: an immune electron microscopic study. 176 13

Ten male goats and five rams were examined from 11 and 15 weeks of age, respectively, for six months to study the ultrasonic appearance of normal testes and epididymides before and after puberty. Five adult rams with lesions of these organs were also examined. A portable, B-mode, real time scanner fitted with a 7.5 MHz, linear array transducer was used. The testis appeared as a homogeneous and moderately echogenic structure with a centrally located mediastinum testis represented by an hyperechogenic line in images taken in the longitudinal plane and by an almost circular spot in transverse images. The testicular capsule and skin were evident as a distinct hyperechogenic line encircling the testicular parenchyma. A thin non-echogenic layer of fluid, presumably between two layers of tunica vaginalis, was observed. The tail of the epididymis was more heterogeneous and less echogenic than the testis. The epididymal head was also less echogenic but homogeneous in texture, and the body of the epididymis was difficult to image. The pampiniform plexus was easily identified as numerous convoluted sonolucent tubular structures. The ultrasonic images of possible cases of epididymitis, spermatocele, testicular cyst and abscess and scrotal hernia are described.
Vet Rec 1991 May 25
PMID:B-mode real time ultrasonographic imaging of the testis and epididymis of sheep and goats. 186 75

The distribution of actin in spermatogenic cells and epididymal spermatozoa of the opossum, Monodelphis domestica, was examined by immunofluorescence microscopy to identify its potential function in the major structural events of sperm development. In spermatogenic cells actin was located at the site of initial interaction between the nucleus and acrosome and remained present through subsequent acrosome morphogenesis. Actin was also associated both with the posterior pole of the nucleus, at the site of flagellar attachment, and with the manchette. Thus actin may play a role in establishing the specific associations of spermatid organelles and in the streamlining of the cells' architecture. In epididymal spermatozoa two sites of actin localization are present. The first site is surrounding the connecting piece where it may participate in the characteristic 90 degrees rotation of the head. The second site was a ring of actin surrounding the lateral boundary of the acrosome where it may play a role in the sperm pairing process which also occurs in the epididymis.
Anat Rec 1991 Jun
PMID:Changes in actin distribution during sperm development in the opossum, Monodelphis domestica. 186 97

Using specific polyclonal antibodies generated against a 13 KD human testicular inhibin, immunocytochemical localization of inhibin was carried out in different regions of human epididymis. The concentrations of inhibin were greater in caput and corpus regions as compared to the caudal region. The epididymal inhibin was found to be bioactive, since it suppressed specifically the FSH levels of rat pituitaries in vitro. Spermiophage/macrophage cells exhibited strong staining for inhibin which were suggestive of a possible role of inhibin in modulation of immune function. In view of the known activities of inhibin in cellular growth, differentiation, and steroidogenesis, epididymal inhibin could have a role in acquisition of sperm fertilizing capabilities.
Anat Rec 1991 Aug
PMID:Occurrence of bioactive and immunoreactive inhibin (13 KD) in human epididymis. 192 51

The distribution of Calmodulin was examined during spermiogenesis and sperm epididymal maturation in rabbit, hamster, mouse, rat, monkey, and human. An affinity-purified antibody to Calmodulin was used to characterize this protein in sperm extracts by immunoblot analysis. Post-embedding immunogold procedures were used to localize Calmodulin at the ultrastructural level. The pattern of Calmodulin distribution was similar in the six species studied. A diffuse labeling was observed in round spermatids. Gold particles accumulated first in the subacrosomal layer of elongating spermatids. The perinuclear ring was also labeled. During the maturation phase of spermatids, Calmodulin labeling extended to the postacrosomal sheath. Dramatic changes occurred at spermiation so that in testicular sperm Calmodulin immunostaining was predominant in the postacrosomal sheath. Some labeling was still detected in restricted areas of the subacrosomal layer. This feature varied from species to species. Calmodulin location did not change during sperm epididymal maturation. A role for Calmodulin in the control of manchette development and regulation of subacrosomal actin aggregation state during spermiogenesis is proposed. The unique location of Calmodulin in the postacrosomal sheath of all species that have been studied in this work, together with the known presence of calcium in this area suggest a pivotal role for Calmodulin in sperm-egg fusion process.
Anat Rec 1991 Aug
PMID:Localization of calmodulin in perinuclear structures of spermatids and spermatozoa: a comparison of six mammalian species. 192 53


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