Seasonal Changes in the Immunolocalization of Cytoskeletal Proteins and Laminin in the Testis of the Black-Backed Jackal (Canis mesomelas).

Manipulation of the reproductive activity of jackals is dependent on a thorough understanding of the reproductive biology of this species. This study describes seasonal morphological changes in the adult testis of the black-backed jackal in relation to the immunoexpression of the basement membrane marker, laminin and the cytoskeletal proteins, cytokeratin, smooth muscle actin and vimentin. Laminin was immunolocalized in basement membranes surrounding seminiferous tubules, as well as in basement membranes associated with Leydig, peritubular myoid and vascular smooth muscle cells. Scalloped basement membranes enclosed seminiferous tubules in regressing testes. The seminiferous epithelium and interstitial tissue in all animals studied were cytokeratin immunonegative. Smooth muscle actin was demonstrated in vascular smooth muscle cells, as well as in peritubular myoid cells encircling seminiferous tubules. Vimentin immunoreactivity was exhibited in the cytoplasm of Sertoli cells, Leydig cells, vascular endothelial cells, vascular smooth muscle cells and fibrocytes. Vimentin immunostaining in Sertoli, Leydig and peritubular myoid cells varied depending on the functional state of the testis. The results of the study have shown that dramatic seasonal histological changes occur in the testes of the jackal. In addition, the use of immunohistochemistry accentuates these morphological changes.

Immunolocalization of intermediate filaments and laminin in the oviduct of the immature and mature Japanese quail (Coturnix coturnix japonica).

This study describes the distribution of vimentin, desmin, smooth muscle actin (SMA) and laminin in the oviduct of the immature and mature Japanese quail. The cytoskeletal proteins vimentin, desmin and SMA have been shown to be involved in cellular support, differentiation, migration and contractility. Laminin is a major component of basement membranes. Luminal epithelia in the infundibular and magnal regions of immature and mature birds exhibited strong vimentin immunoreactivity. Luminal epithelial cells exhibiting strong vimentin immunoreactivity were present in the isthmus and shell gland regions of only mature quails. Infundibular glandular grooves displayed strong vimentin immunostaining. In contrast, the glandular epithelia of the magnum, isthmus and shell gland were vimentin immunonegative. Fibroblasts and vascular endothelial cells in the lamina propria of the oviductal regions studied exhibited strong vimentin immunostaining. Smooth muscle cells forming the tunica muscularis and vascular tunica media displayed strong desmin and SMA immunostaining. Strong laminin immunostaining was demonstrated in the basement membranes associated with smooth muscle cells, as well as in the basement membranes underlying the luminal and glandular epithelia. In conclusion, this study has shown that the immunolocalization of desmin, SMA and laminin in the oviduct of the Japanese quail is similar to that in the domestic fowl. However, differences in the immunoexpression of vimentin in the LE of the two avian species were shown to exist. In addition, the study has shown that the immunolocalization of vimentin in the Japanese quail varies depending on the oviductal region, as well as the developmental stage of the oviduct.

Post-hatch changes in the immunoexpression of desmin, smooth muscle actin and vimentin in the testicular capsule and interstitial tissue of the Japanese quail (Coturnix coturnix japonica).

The post-hatch development of immunoreactivity to desmin, smooth muscle actin (SMA) and vimentin in the testicular capsule and interstitial tissue of day-old to adult quails was described in this study. The tunica albuginea of the testicular capsule was composed mainly of myoid cells. A zonal arrangement of desmin and SMA immunostaining was observed in myoid cells of the tunica albuginea in 1- to 24-day-old quails. Immunostaining for SMA and desmin was uniform in the tunica albuginea of adult birds. Vimentin immunostaining in the testicular capsule was demonstrated in mesothelial cells, endothelial cells and fibroblasts. The interstitial tissue contained mesenchymal cells, peritubular myoid cells, Leydig cells and fibroblasts. Desmin-immunopositive mesenchymal cells were present in the interstitial tissue of 1- to 17-day-old quails. Peritubular myoid cells expressed strong desmin immunostaining in all developmental stages, while the intensity of SMA immunostaining increased with testicular maturation. Vimentin was demonstrated in Leydig cells and fibroblasts, while the peritubular myoid cells displayed strong vimentin immunostaining only in adult birds. Strong vimentin immunostaining was demonstrated in the endothelial cells of capsular and interstitial blood vessels. The tunica media of these blood vessels displayed desmin and SMA immunostaining. The results of the study have established that variability exists in the distribution and intensity of desmin, SMA and vimentin immunostaining in the testicular capsule and interstitial tissue of the post-hatch Japanese quail.

An immunohistochemical study of the oviduct in the domestic fowl (Gallus domesticus).

This study describes the distribution of vimentin, desmin, smooth muscle actin (SMA) and laminin in the oviduct of the laying domestic fowl. Vimentin immunostaining was localised in the luminal epithelium of the infundibulum, magnum, magnum-isthmus junction and isthmus. The luminal epithelium of the shell gland regions displayed weak vimentin immunostaining. Vimentin immunostaining was demonstrated in the glandular grooves of the tubular infundibular region. In contrast, gland cells in the magnum, isthmus and shell gland regions were vimentin immunonegative. Fibroblasts and vascular endothelial cells in the lamina propria of the oviductal regions studied exhibited vimentin immunostaining. Strong desmin and SMA immunostaining were present in the smooth muscle cells of the tunica muscularis and vascular tunica media. In this study, basement membranes underlying the luminal and glandular epithelia were immunopositive for laminin. In addition, basement membranes associated with smooth muscle cells exhibited laminin immunostaining. The results of the study indicate that the immunolocalisation of desmin, SMA and laminin in the oviduct of the domestic fowl is similar to that in the mammalian uterus. The immunolocalisation of vimentin in the domestic fowl varies depending on the oviductal region.

An immunohistochemical study of ovarian follicle histogenesis in the early post-hatch Japanese quail (Coturnix coturnix japonica).

The early post-hatch development of immunoreactivity to vimentin, desmin, smooth muscle actin (SMA) and laminin, in relation to follicle histogenesis, was described in this study. Ovigerous cords in day old quails contained pre-granulosa cells and oocytes. Pre-granulosa cells at the cortico-medullary junction were vimentin immunopositive. A laminin immunopositive basement membrane and desmin immunopositive mesenchymal cells lined the ovigerous cords. Ovigerous cords in 3-day-old quails contained developing primordial follicles, the vimentin immunopositive pre-granulosa cells of which were partially encircled by a basement membrane and desmin immunopositive mesenchymal cells. In 5- to 7-day-old quails, ovigerous cords formed an outer cortical region, while primordial follicles formed the inner cortical region. Early pre-vitellogenic follicles were present in 9- to 13-day-old quails. Underlying the granulosa cells of these follicles was a laminin immunopositive basement membrane and a layer of desmin immunopositive thecal cells. Early and late pre-vitellogenic follicles dominated the ovary in 15- to 17-day-old quails. The thecal layer in these follicles was desmin immunopositive, but SMA immunonegative. The results of the study have shown that the process of primordial follicle development in the Japanese quail is similar to that reported in mammals. The study suggests that in the quail pre-granulosa cells originate predominantly from the medulla. The study has shown that, in the Japanese quail, thecal cells are derived from desmin immunopositive mesenchymal cells lining the ovigerous cords.

The excurrent ducts of the testis of the emu (Dromaius novaehollandiae) and ostrich (Struthio camelus): Microstereology of the epididymis and immunohistochemistry of its cytoskeletal systems.

The volumetric proportion of the various ducts of the epididymis of the emu and ostrich and the immunohistochemistry of actin microfilaments, as well as cytokeratin, desmin and vimentin intermediate filaments, were studied in the various ducts of the epididymis of the emu and ostrich. The volumetric proportions of various ducts, which are remarkably different from those of members of the Galloanserae monophyly, are as follows: the rete testis, 5.2 +/- 1.4% for the emu and 2.4 +/- 1.8% for the ostrich; efferent ducts, 14.2 +/- 2.3% (emu) and 11.8 +/- 1.8% (ostrich); epididymal duct unit, 25.8 +/- 5.8% (emu) and 26.1 +/- 4.1% (ostrich) and connective tissue and its content, 54.7 +/- 5.8% (emu) and 60.0 +/- 4.9% (ostrich). Unlike in mammals and members of the Galloanserae monophyly, only vimentin was immunohistochemically demonstrated in the rete testis epithelium of the emu, and none of the cytoskeletal protein elements in the ostrich rete testis. The epithelium of the efferent ducts of the emu co-expressed actin, cytokeratin and desmin in the non-ciliated type I cells, and vimentin in the ciliated cell component. The ostrich demonstrated only cytokeratin in this epithelium. The ratite epididymal duct unit is different from that of mammals in lacking actin (only weaky expression in the ostrich), desmin and cytokeratin, and a moderate/strong immunoexpression of vimentin in the basal cells and basal parts of the NC type III cell in the epididymal duct unit. Immunoexpression of the microfilaments and intermediate filaments varied between the two ratite birds, as has been demonstrated previously in birds of the Galloanserae monophyly, and in mammals.

The immunohistochemical localization of desmin and smooth muscle actin in the ovary of the African giant rat (Cricetomys gambianus) during the oestrous cycle.

The aim of this study was to describe the distribution of smooth muscle actin and desmin immunopositive cells in the ovary of the giant rat. In addition, the study describes the morphological changes in the ovary of this species during the oestrous cycle. Healthy secondary and tertiary follicles dominated the ovary during pro-oestrus and oestrus. The theca externa of the tertiary follicles was immunopositive for smooth muscle actin, but immunonegative for desmin. Oestrus was also characterized by the presence of corpora haemorrhagica, which had an outer layer of smooth muscle actin immunopositive cells. Differentiating corpora lutea were observed during metoestrus. A further notable feature of the ovary during metoestrus was the presence of numerous atretic secondary and tertiary follicles. In the later stages of atresia, the follicles were infiltrated by desmin and smooth muscle actin immunopositive cells. Dioestrus was characterized by the presence of non-regressing and regressing corpora lutea. Immunostaining for smooth muscle actin was demonstrated in the enclosing layer of the corpora lutea, as well as in the tunica media of blood vessels within the corpora lutea. The results of this study have shown that morphological changes in the ovary of the giant rat during the oestrus cycle are similar to those of laboratory rodents. Furthermore, the results of the immunohistochemical study indicate that the perifollicular distribution of desmin and smooth muscle actin cells changes during follicular development and atresia.

Immunohistochemical localization of the progesterone and oestrogen alpha receptors in the uterine horns of the African giant rat (Cricetomys gambianus).

The present study investigated the immunolocalization of the progesterone and oestrogen alpha receptors in the uterine horns of the African giant rat during the oestrous cycle. The progesterone and oestrogen alpha receptors were demonstrated in various cellular constituents of the endometrium, myometrium and perimetrium. The intensity of progesterone and oestrogen alpha receptor immunostaining in the endometrial and myometrial layers of the uterine horns varied during the oestrous cycle. The intensity of oestrogen alpha receptor immunoreactivity in the luminal epithelium was high during pro-oestrus, oestrus and dioestrus. Progesterone and oestrogen alpha receptor immunoreactivity in the endometrial epithelia was absent during metoestrus. Moderate to strong immunostaining for the progesterone and oestrogen alpha receptors was demonstrated in the myometrial smooth muscle cells during pro-oestrus, oestrus and dioestrus. The intensity of progesterone and oestrogen alpha receptor immunostaining in the myometrial smooth muscle cells was low during metoestrus. Stromal cells in the perimetrium consistently expressed progesterone and oestrogen alpha receptor immunoreactivity throughout the oestrous cycle. The findings of the study indicate that in the giant rat the immunolocalization of the progesterone and oestrogen alpha receptors, in endometrial and myometrial regions of the uterine horns, varies during the oestrous cycle.

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.

An immunohistochemical study of ovarian innervation in the emu (Dromaius novaehollandiae).

The present study investigated the distribution of nerves in the ovary of the emu. The neuronal markers, protein gene product 9.5, neurofilament protein and neuron specific enolase demonstrated the constituents of the extrinsic and intrinsic ovarian neural systems. The extrinsic neural system was composed of ganglia in the ovarian stalk, as well as nerve bundles, which were distributed throughout the ovary. Isolated neuronal cell bodies, in the medulla and cortex, formed the intrinsic neural system. An interesting finding of the study was the presence of nerve bundles, circumscribed by lymphocytes, in the ovarian stalk. The findings of the study indicate that the distribution of nerve fibres and neuronal cell bodies in the emu ovary is similar, but not identical to that of the domestic fowl and ostrich.

Ultrastructural features of atretic follicles in the sexually immature ostrich (Struthio camelus).

The aim of this study was to describe the ultrastructural features of atresia in follicles of the immature ostrich (12-14 months old); a ratite that displays seasonal, precocious ovarian activity. The early stage of atresia in primordial, pre-vitellogenic and vitellogenic follicles was characterized by the accumulation of lipid droplets in the granulosa cells. Granulosa cells with condensed cytoplasm and nuclei were a prominent feature during the intermediate phase of atresia. The degenerating follicles were then infiltrated by stroma during the terminal stages of atresia. The results of this study provide further information on the morphology of atretic ovarian follicles in the immature ostrich.

Morphological and immunohistochemical study of testicular capsule and peritubular tissue of emu (Dromaius novaehollandiae) and ostrich (Struthio camelus).

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 microm for the free surface of the ostrich testis, and 176.2+/-57.5 microm 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.

An ultrastructural characterization of the ooplasm in ovarian follicles of the immature ostrich (Struthio camelus).

Primordial, pre-vitellogenic and vitellogenic follicles were present in the ovary of the immature ostrich. The oocytes of these follicles were composed of a nucleus surrounded by ooplasm. Central, intermediate and cortical regions formed the ooplasm. The organelles present in these ooplasmic regions varied depending on the stage of follicular development. In primordial and small pre-vitellogenic (100-150 microm in diameter) follicles the central region of the ooplasm was dominated by an accumulation of organelles, which formed Balbiani's vitelline body. In contrast, the central region in vitellogenic follicles was filled with numerous large yolk spheres, many of which contained lining bodies. Numerous lipid droplets interspersed with mitochondria and small yolk spheres formed the intermediate ooplasmic region in primordial and small pre-vitellogenic follicles. In large pre-vitellogenic (150-400 microm in diameter) and vitellogenic follicles the intermediate region contained a greater density of mitochondria and small yolk spheres. Small yolk spheres were observed in the cortical region of pre-vitellogenic follicles. An interesting feature of the cortical region in vitellogenic follicles was the frequent occurrence of Golgi complexes. The results of the study indicate that although the ovarian follicles in the immature ostrich are not ovulated, the components and composition of the ooplasm are similar to those observed in the mature follicles of other avian species.

Ultrastructural features of the uterus in the sexually immature ostrich (Struthio camelus) during periods of ovarian inactivity and activity.

The ultrastructure of the surface epithelium and tubular glands of the uterus in the immature ostrich is described. In ostriches with inactive ovaries the uterus is lined by a non-ciliated simple columnar epithelium, with basally located heterochromatic nuclei. Scanning electron microscopy revealed that these non-ciliated cells have a dense microvillous cover. A simple columnar to pseudostratified columnar epithelium, comprised of non-ciliated and ciliated cells, lines the uterus in birds with active ovaries. The ciliated cells possess a wide luminal region, which contains a nucleus and various organelles. An accumulation of secretory granules was observed in the apical regions of the non-ciliated cells, as well as in a few ciliated cells. In addition to non-ciliated and ciliated cells, a cell type with rarefied cytoplasm was also identified. These cells appear to correspond to calcium secreting cells identified in other avian species. The results of this study indicate that, although uterine differentiation is present in immature ostriches with active ovaries, the production of secretory product appears to occur mainly in non-ciliated epithelial cells.

An immunohistochemical study of the distribution of intermediate filaments in the ovary of the emu (Dromaius novaehollandiae).

The immunohistochemical localization of the intermediate filaments desmin, vimentin and smooth muscle actin (SMA) in the ovary of the emu was described in the present study. The cortical region of the ovary contained developing and atretic primordial, pre-vitellogenic and vitellogenic follicles. Vimentin immunostaining was demonstrated in the granulosa cell layer of primordial, pre-vitellogenic and vitellogenic developing and atretic follicles. An interesting finding of the present study was the localization of SMA in fibroblasts located in the theca externa of late vitellogenic follicles. The presence of SMA in these fibroblasts suggests that they possess characteristics of smooth muscle cells.

Ultrastructural features of the follicular wall in developing follicles of the sexually immature ostrich (Struthio camelus).

The ultrastructure of the follicular wall in primordial, previtellogenic and vitellogenic follicles of the sexually immature ostrich is described in the present study. The follicular wall consists of a zona radiata, granulosa cell layer, basal lamina and thecal layer. Cytoplasmic processes from the plasma membranes of the granulosa cell layer and the ovocyte form the zona radiata in previtellogenic and vitellogenic follicles. The granulosa cell layer transforms from simple cuboidal epithelium in primordial follicles to simple columnar or pseudostratified columnar epithelium in previtellogenic and vitellogenic follicles. Transosomes were observed along the apical and lateral plasma membranes of granulosa cells. The thecal layer in previtellogenic and vitellogenic follicles consists of interna and externa components. The fibroblasts in the theca externa contain microfilaments, which are thought to be actin filaments. The study revealed ultrastructural features, which are associated with the transportation of yolk precursors and nutrients into the ovoplasm. In addition, the study indicates that, although the cells in the theca externa contain microfilaments, they do not possess the ultrastructural characteristics of smooth muscle cells.

Immunoreactivities to protein gene product 9.5, neurofilament protein and neuron specific enolase in nerves in the oviduct of the sexually immature ostrich, Struthio camelus.

The present study investigated the distribution of nerves in the oviduct of the sexually immature ostrich. The presence of protein gene product 9.5, neurofilament protein and neuron specific enolase nerve fibres were demonstrated in the infundibulum, magnum, isthmus, shell gland and vagina. Nerve fibres containing protein gene product 9.5, neuron specific enolase and neurofilament protein were particularly numerous in the tunica muscularis and intermuscular connective tissue areas of the shell gland and vagina. The presence of a large number of nerves in these oviductal regions is probably important in the coordination of muscle contraction. An interesting finding of the study was the presence of protein gene product 9.5 and neuron specific enolase immunopositive nerve fibres in the walls of blood vessels. It is likely that these nerves are autonomicin nature and play a role in the regulation of blood flow. This study has shown the presence of an extensive neural network in the oviduct of the ostrich. In addition, the results of the investigation have shown that the neuronal markers protein gene product 9.5, neurofilament protein and neuron specific enolase can be used to demonstate nerve fibres in the ostrich.

A morphological and immunohistochemical study of healthy and atretic follicles in the ovary of the sexually immature ostrich (Struthio camelus).

The morphology of healthy and atretic follicles in the ovary of the sexually immature ostrich was described in the present study. In addition, the distribution of the intermediate filaments desmin, vimentin and smooth muscle actin, in these ovarian follicles, was demonstrated. Healthy and atretic primordial, pre-vitellogenic and vitellogenic follicles were present in the ovaries of the sexually immature ostrich. Atresia occurred during all stages of follicular development. Atretic primordial and pre-vitellogenic follicles were characterized by the presence of a shrunken oocyte surrounded by a multilayered granulosa cell layer. Two forms of atresia (types 1 and 2) were identified in vitellogenic follicles. In the advanced stages of type 1 atresia the follicle was dominated by a hyalinized mass. In contrast, in type 2 atresia the granulosa and theca interna cells differentiated into interstitial gland cells. Positive immunostaining for desmin was observed in the granulosa cells of only healthy primordial and pre-vitellogenic follicles. Atretic primordial and pre-vitellogenic follicles were immunonegative for desmin. Vimentin immunoreactivity was demonstrated in the granulosa cells of all follicles except the vitellogenic atretic follicles. The results of the present study indicate that ovarian follicles in the sexually immature ostrich undergo a cycle of growth and regression, which is similar to that reported in other avian species. Furthermore, based on the results of the immunohistochemical study, it would appear that the distribution and immunostaining of intermediate filaments changes during follicular development and atresia.

Immunoreactivities to protein gene product 9.5, neurofilament protein and neuron specific enolase in the ovary of the sexually immature ostrich (Struthio camelus).

The innervation of the ovary has been studied in various species of birds and mammals. Despite the fact that the innervation of any organ is an essential factor in controlling its growth and function, no information is available on the distribution of nerve fibers in the ovary of the sexually immature ostrich. Thus, the present study was undertaken to investigate the distribution of nerve fibers in the ovary of the sexually immature ostrich, using antibodies against neurofilament protein type M of 160 kD (NP), protein gene product 9.5 (PGP 9.5) and neuron specific enolase (NSE). A total of 26 sexually immature female ostriches, aged between 12 and 14 months were used in the present study. Immunostaining was performed using a LSAB plus kit (Dakocytomation, Denmark). Antibodies against NP and PGP 9.5 were used at dilutions of 1:25 and 1:50, respectively. A ready-to-use solution containing antibodies against NSE was also used. Strong immunostaining for NP, PGP 9.5 and NSE was observed in nerve bundles, which coursed through the ovarian stalk and extended into the medulla and cortex. In addition, NSE immunoreactive nerve cell bodies were observed in the cortex and medulla. NP, PGP 9.5 and NSE immunoreactive nerve fibers were present in the thecal layer of the follicular wall. The current study has highlighted the distribution of NP, PGP 9.5 and NSE-immunoreactive nerve fibers in the ovary of the sexually immature ostrich. The findings of the present study suggest that the distribution of nerve fibers in the immature ostrich is similar to that of the domestic fowl.

Morphological features of the luminal surface of the magnum in the sexually immature ostrich (Struthio camelus).

Summary Observations were made, using scanning electron microscopy, of the surface features of the magnum in the immature ostrich during periods of ovarian inactivity, activity and regression. In birds with inactive ovaries the luminal surface of the magnum was lined with non-ciliated cells, which were densely covered by microvilli. In contrast, the magnum in birds with active ovaries was composed of ciliated and non-ciliated cells. The distribution of ciliated cells was not uniform, with clumps of cilia occurring next to non-ciliated areas. Samples collected from birds with regressing ovaries, during periods of decreasing daylength, revealed that the magnum was undergoing involution. The deciliation of ciliated cells and the presence of short microvilli on non-ciliated cells characterized magnal regression. These results suggest that ovarian activity and changes in daylength have a profound effect on the surface features of the magnum in the immature ostrich.