Morphology, development and comparative anatomical evaluation of the testicular excretory pathway in Acipenser.

In the differentiated state, the testicular excurrent duct system of the sturgeon begins as a longitudinal marginal network of the testis, extending along the entire length of the male gonad. From here, mesorchial transversal ducts travel to the ventral aspect of the pars sexualis of the opisthonephros where they merge behind the dorsal coelomic wall to form the longitudinal marginal network of the kidney. Then, the seminal pathways enter the confines of the pars sexualis of the opisthonephros and divide into a complicated, multipartite system consisting of (1) centropapillary ducts, situated in the center of a group of urinary collecting ducts, (2) lacunary basal sinuses, located on the bases of opisthonephric columns and (3) intracolumnar ducts running inside the renal columns, the latter representing typical functional units of the adult sturgeon kidney. The contacts between intracolumnar ducts and the vascular poles of corresponding renal corpuscles represent the urogenital junction in the sturgeon. The nephrons of the pars sexualis involved in sperm transport do not lose their urinary functions, but are histologically identical to those of the pars excretoria which are solely urinary. The opisthonephros of sturgeons grows continuously by the formation of new nephrons from an opisthonephric blastema located on the base of each renal column. A close topographical association between this blastema tissue and the lacunary basal sinuses of the testicular excurrent duct system guarantees that new renal corpuscles in the pars sexualis are included in the seminal passage from their beginning. From the urogenital junctions, on their way to the exterior, the spermatozoa have to travel through Bowman's capsules and tubules of the nephrons involved, then through the urinary collecting ducts, the wolffian duct and finally the sinus urogenitalis. The development of the testicular excurrent duct system begins in 8-month-old animals in the pregonadal area of the gonadal fold. Here, a primary gonoductal blastema proliferates to form a longitudinal network of anastomosing strands, situated in the dorsal mesogonadal attachment. From this primary longitudinal network, small tubules grow into the direction of the opisthonephros and into the direction of the testis. In the period from 8 to 18 months, the testicular excurent duct system reaches the adult state. In conclusion, the testicular excurrent ducts of sturgeons initially develop similar to those of Polypterus and in modern teleosts from a primary longitudinal system, beginning in the pregonadal area, localized in the mesogonadal attachment and extending caudally. Then, in a second step of development, the phylogenetically older situation, using parts of the kidney as passage, already seen in Chondrichthyes, but preserved also in higher vertebrates, is achieved in Acipenser. For this, seminal ducts grow into the opisthonephros and establish here the urogenital junctions with corresponding renal corpuscles. Furthermore, the initially longitudinally oriented ducts in the mesogonadal attachment partly lose their continuity and become integrated into the course of the transversal mesorchial ducts, represented by their portions with the widest lumina and the thickest walls.

Histochemical in situ identification of bovine embryonic blood cells reveals differences to the adult haematopoietic system and suggests a close relationship between haematopoietic stem cells and primordial germ cells.

Cryostat sections of bovine embryos of exactly known age (obtained from artificial insemination), ranging from 32 to 60 days post-insemination, were treated with a wide range of antibodies directed against cell surface antigens or lineage-specific factors in order to demonstrate different types of fetal blood cells and their precursors. An antibody specific to bovine c-kit (bk-1) stained not only presumptive haematopoietic stem cells in the dorsal aorta and the embryonic liver, but also a subpopulation of putative primordial germ cells in the gonadal anlage, the latter being further characterised by a positive labelling with the lectins STA, WFA and WGA and a histochemical reaction for alkaline phosphatase. The antibody against CD 45, commonly regarded as a pan-leukocyte marker, reacted in the bovine embryo with different types of blood cells, as well as with presumptive vasculogenetic cells and a subpopulation of putative primordial germ cells. CD 61 immunoreaction proved to be a useful tool for demonstrating megakaryocytopoiesis in the embryonic liver, in addition to the lumen of blood vessels and the mesonephros. Staining with BM-2 was restricted to a single population of medium-sized, round to oval cells, forming small groups within the parenchymal strands of the liver. Characterised furthermore by a U-shaped nucleus, this BM-2-positive cell type apparently represents a developmental stage in the granulopoietic lineage. B-lymphocytopoiesis in the bovine liver was detected with antibodies directed against WC-4 and IgM, but not until day 58 post-insemination. Using antibodies to CD 14, no positive results could be obtained in embryonic tissues, although anti-CD 14-positive macrophages were easily recognised in lymph nodes of adult bovines. The antibody against CD 68, however, identified two populations of primitive macrophages in our samples. One population was located in parenchymal strands of the embryonic liver, probably acting as nursing cells for haematopoietic foci, and the other was observed intravasally in the sinusoids of the liver, most probably representing primitive Kupffer cells.

The genus Acipenser as a model for vertebrate urogenital development: the müllerian duct.

The development of the müllerian duct was studied in a total of 85 specimens of Acipenser ruthenus and Acipenser baeri in the period from 7 days after hatching through 5 years of age. Normal histology on serial sections, transmission and scanning electron microscopy and bromodeoxyuridine immunohistochemistry were applied. In Acipenser, the primary set of opisthonephric nephrons possess short nephrostomial tubules with well-developed nephrostomes. Proliferating cells from the lateral side of the slightly protruding nephrostomial lips spread out over the ceolomic surface, replace here the flat mesothelium of lateral plate origin and establish the infundibular field, consisting of cuboidal or columnar cells. At about 28 days after hatching, the primordium of the müllerian infundibulum becomes visible in the form of a pocket-like invagination within the infundibular field. This invagination is found coexisting with and located laterally to the line of intact nephrostomes. The müllerian infundibulum, therefore, does not represent the homologon of a nephrostome itself, but must be regarded as a separate and secondary structure. The müllerian duct proper has its origin in cells from the bottom of the infundibular pocket. These cells grow as a tubule with a solid tip in the caudal direction, paralleling the wolffian duct, but without a contribution of cells from the latter. In Acipenser, a müllerian duct is present also in the adult male. In males as in females, the caudal extremity of the müllerian duct generally divides into two to three smaller terminals which end in the wolffian duct at different levels, but always cranial to the urogenital sinus. In most indifferent animals and in all males of this study, the tips of the müllerian terminals are closed and covered by a thin layer of wolffian epithelium. In adult females, the müllerian ducts end with open terminals. In both sexes, the adult müllerian duct is lined by a pseudostratified columnar epithelium, consisting of ciliated, non-ciliated and basal free cells.

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 anti-sera 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.

The genus Acipenser as a model for vertebrate urogenital development: ultrastructure of nephrostomial tubule formation and of initial gonadogenesis.

The ultrastructure of the nephrostomial tubule and the gonadal crest of Acipenser ruthenus were studied by transmission and scanning electron microscopy. Opisthonephric nephrostomial tubules begin to appear at the end of the first week after hatching and are regularly segmentally arranged and completely developed in the period between 10 and 35 days after hatching. Fully differentiated nephrostomial tubules connect the vestibular area of the Malpighian corpuscle with the coelomic cavity as short, open canals. Before reaching the latter, each nephrostomial tubule widens to a funnel-like structure and ends with a round opening, the nephrostome. The outer rim of the nephrostome protrudes slightly into the coelomic cavity forming the nephrostomial lips. The nephrostomial tubule is lined by one layer of cuboidal or low-columnar cells, equipped with an apical set of long kinocilia, which point generally in the direction of the Malpighian corpuscle. The cilia of the distal funnel region, however, point toward the coelomic cavity. The cells of the nephrostomial lips preserve a lower degree of cellular differentiation than the rest, display a blastema-like appearance and proliferate by frequent mitoses. Proliferating cells of the nephrostomial lips spread out on the coelomic surface and replace the flat mesothelium of lateral plate origin here. Furthermore, cells of the nephrostomial lips also show the tendency to grow downward, giving the epithelium of the lip region a multilayered appearance. Proliferating cells of the medial nephrostomial lips associate with accumulations of germ cells and form the primordium of the gonad (gonadal crest). Within the crest, the intercellular space between the germ cells and the surrounding epithelial supporting cells is filled with a basal lamina-like, electron-dense substance and may contain short, interlocking processes of the two cell types. The large germ cells of this early period have lobulated, electron-lucent nuclei, spherical mitochondria with inclusions and abundant narrow profiles of the smooth endoplasmatic reticulum.

On the intrinsic innervation of the epididymis of the camel (Camelus dromedarius).

The innervation of the camel epididymis was studied in 26 apparently healthy, sexually mature animals aged between 4 and 12 years. The material was collected during the different seasons of the year. Generally, five samples were taken from each epididymis. To demonstrate the general innervation pattern, immunohistochemical reactions to protein gene product-9.5, neurofilaments and neuron-specific enolase were used, in addition to acetylcholinesterase histochemistry. The nerve supply of the epididymis comes from two sources: (1) The majority of fibers come from the N. spermaticus inferior and accompany the deferent duct. (2) Another contribution stems from the N. spermaticus superior and enters the head region of the epididymis. From the exterior, the nerves penetrate the capsule of the organ to reach the interductular connective tissue. The terminal ramifications are observed directly within the wall of the duct and the wall of the epididymal arteries. The veins of the camel epididymis are not innervated. In the wall of the ductus epididymidis, the nerve fibers form plexuses at the subepithelial level and in the muscular coat. The amount of nerve fibers increases from the head to the tail, paralleling an increase in the intrinsic musculature. The intramural and interductular innervation of epididymal body and tail shows clear seasonal variations: More fibers and stronger reactions are observed during the winter season; the lowest density and the weakest reactions occur during the summer season. All epididymal nerves of the camel are unmyelinated. The majority of the intramural fibers and all in the arterial wall represent postjunctional sympathetic axons, but in the intramural plexuses of the duct a considerable number of cholinergic fibers are also present. Neuropeptide Y is the most frequent peptidergic transmitter and generally co-localized with dopamine-beta-hydroxylase in the sympathetic axons. Vasoactive intestinal polypeptide has a distribution similar to that of the cholinergic fibers. Calcitonin gene-related peptide-positive axons occur in moderate numbers, but never in the arterial innervation. Together with the relatively rare substance P-containing fibers, the calcitonin gene-related peptide-positive axons seem to represent the only sensory nerves in the camel epididymis.

Immunohistochemical investigations of the autonomous nerve distribution in the testis of the camel (Camelus dromedarius).

The distribution of autonomous nerves in the testis of the camel was studied by immunohistochemical methods. A total of 26 testes was collected during the different seasons of the year. As pan-neuronal markers, antibodies to protein gene product 9.5 and to neurofilaments are superior to antibodies against neuron-specific enolase and acetylcholinesterase histochemistry for the description of the nerves in the camel testis. Testicular nerves reach the camel testis by three access-routes as (1) funicular contribution, (2) mesorchial contribution and (3) as caudal contribution. The main target for testicular nerves is the arterial vascular tree of the organ, whereas all veins of testis and pampiniform plexus are devoid of any innervation in the camel. In the wall of the arteries, the nerves form a plexus at the media-adventitia border. The density of the arterial plexuses increases along the vascular tree: smaller septal and mediastinal arteries are better innervated than albugineal arteries and the latter better than the A. testicularis. The nerves in the septula testis, in the mediastinum and between the Leydig cells show clear seasonal changes, being particularly abundant in autumn and particularly scarce in spring. The nerves that reach the camel testis are unmyelinated and represent in the vast majority postjunctional sympathetic neurons. Cholinergic fibers are absent in the camel testis. Neuropeptide Y is the dominating peptidergic transmitter in the testicular nerves and colocalized with noradrenaline in the same axons. Vasoactive intestinal polypeptide-containing fibers reach the camel testis exclusively as parts of the caudal nervous contribution via the ligamentous bridge between testis and epididymal tail and are restricted to the caudal pole of the testis. Calcitonin gene-related peptide-positive axons are not frequent in the camel testis; nevertheless, they seem to be the most important sensory pathway of this organ.

The genus Acipenser as a model system for vertebrate urogenital development: nephrostomial tubules and their significance for the origin of the gonad.

Early gonadal development was studied in the sterlet, Acipenser ruthenus, by means of histological and semithin serial sections and scanning electron microscopy. Special attention was given to the role of opisthonephric nephrostomial tubules and their coelomic funnels (nephrostomes, coelomostomes) in the origin of the gonad. Specimens of about 1 mm in length (about 7 days post hatching) have a continuous kidney complex (holonephros) that extends from the branchial region to the level of the cloaca and may be divided into a cranial pronephros and a caudally following opisthonephros, with no overlapping of either portion. In specimens of 10 to 25 mm in length the regression of pronephros and cranial opisthonephros can already been seen; as a consequence, these parts of the kidney complex are not involved in gonadogenesis. The initial gonadal anlage is seen in specimens of 30-40 mm in length. The somatic cells of the gonadal primordium develop from the medial lips of segmentally arranged opisthonephric nephrostomes situated in a line that extends from the level of the stomach through that of the spiral valve. The nephrostomes involved in this process belong to the first-order set of nephrons, since nephrons of higher order that arise continuously from blastema cells during further growth of the animals never send nephrostomial tubules to the coelomic surface. The cells of the medial nephrostomial lips proliferate by many mitoses. They grow over and surround the large germ cells that have accumulated on the medial side of the nephrostomes. The proliferating nephrostomial cells are elongated in shape, and their long axes are oriented in the cranio-caudal direction. By their size, shape and arrangement they replace not only the flat, polygonal, mesothelial cells of lateral plate origin on the medial side of the nephrostomes, but also those in the interstices in between. The result is the formation of a continuous gonadal crest situated medially from the nephrostomial line. At 100-130 mm in length, the gonadal crest has reached the stage of a gonadal fold that is attached to the dorsal body wall by a thin mesogonadium. Stroma cells and blood vessels start to invade the gonadal fold. At 240-290 mm in length, parts of the gonadal fold are converted into a fat body, a structure that is typically present in adult sturgeons of either sex. Nephrostomial tubules and their nephrostomes are visible only for a short period in Acipenser ruthenus. They are completely developed in specimens of 25-40 mm in length and start continuous retrogression immediately afterwards. Therefore, they cannot play an important role in excretion. Their only purpose apparently is to function as precursor tissue for other organs, such as the gonad, in establishing a route by which cells of the intermediate mesoderm can gain access to and spread out over the coelomic surface. In conclusion, the observations made in Acipenser prove that this species is a most suitable model to explain the origin of the gonad from opistho/mesonephric nephrostomial tubules in vertebrates with a less-developed or early regressing pronephros. Most mammals, including man, belong to this category.