Role of the mesonephros as a transient haematopoietic organ in the bovine embryo.

During vertebrate embryogenesis, haematopoietic stem cells (HSC) arise in the aorta-gonads-mesonephros (AGM) region. In the present study, we examined serial sections of 22-34 days post-insemination (dpi) bovine embryos, a species with a well-developed and functional mesonephros. We describe the temporo-spatial distribution of presumptive c-kit positive HSC and the occurrence of haematopoietic foci in the mesonephros and fetal liver using specific antibodies directed against haematopoietic cell markers and conventional electron microscopy. In the mesonephros, presumptive HSC were found at 23-24 dpi in the blood stream, in the endothelial lining of the filtering capillaries and in the septal stroma of the cranial part of the mesonephros, the mesonephric giant corpuscle (MGC), suggesting a colonalization via the blood stream from the haematopoietic clusters of the dorsal aorta. From 25 to 30 dpi, presumptive HSC predominate in the septal stroma of the MGC, were they first expand but then decline and disappear following 32 dpi. In parallel, we found ongoing erythropoiesis and myelopoiesis starting in the MGC at 24 dpi and extending during the complete observation period. In the embryonic liver, colonization with presumptive c-kit positive HSC occurs slightly later, at 25 dpi. Active formation of blood cells in the liver increases following 30 dpi. In conclusion, the mesonephros of bovine embryos, in particular its MGC, functions as a primitive haematopoietic organ, temporarily intercalated between extraembryonic erythropoiesis and haematopoiesis within in the fetal liver, thus recapitulating for a short period a phylogenetically old site of blood formation.

Immunophenotyping and spatio-temporal distribution of aortic cell clusters in the bovine embryo.

In the present study the temporal and spatial appearance of aortic cell clusters in bovine embryos is described. Aorta-associated c-kit-positive cell clusters can be observed first in 23 days post inseminationem (dpi) bovine embryos and disappear after 34 dpi. For the first time, it was shown that the immunophenotype of these aortic cluster cells changes during embryonic development. Aortic cell clusters are c-kit+/CD45-/STA-, when they are first detected in the 23 dpi embryo, and acquire a c-kit+/CD45+/STA- phenotype in 27-29 embryos and a c-kit+/CD45+/STA+ immunophenotype in 32-34-day-old specimens. Cell clusters are most prominent in the vicinity of lateral and ventral aortic branches, but rare in omphalomesenteric arteries and absent in Aa. umbilicales. Free c-kit-positive cells in an intravasal position are common, suggesting separation from the clusters in order to colonize subsequent hematopoietic organs, i.e., the liver and the mesonephros. Transmission electron microscopic analysis reveals the existence of primitive desmosomes between the clusters cells and adjacent endothelial cells as well as a fine basal lamina as a demarcation between the cluster cells and underlying mesenchymal cells. Material resembling extracellular matrix is found in large vacuoles in cluster cells of 23 dpi embryos. Immunocytochemistry reveals an intense accumulation of heparan sulfate proteoglycan and collagen IV in the aortic wall at the sites where cell clusters are attached. These observations suggest that the hematopoietic cell clusters induce the formation of a specific microenvironment within the aortic wall.

Morphology and innervation pattern of the feline urogenital junction.

The feline urogenital junction is situated between the extratesticular rete and the spacious initial segments of the efferent ductules. The rete epithelium is cuboidal to low columnar. The rete cells forming the junction rest on a wavy basal lamina, display deep mutual invaginations, possess central nuclei with several infoldings and form a distinct border with the columnar epithelial cells of the initial segments of the ductuli efferentes. The epithelium of the initial segments is composed of ciliated cells and non-ciliated principal cells. The latter are the dominating type and characterized by an apical brush-border and a supranuclear endocytotic apparatus. The stroma of the extratesticular rete contains an abundance of collagen whereas contractile cells are here generally absent. In contrast, the initial segments of the efferent ductules are surrounded by elastic fibres and a layer of contractile cells. All nerves for the feline urogenital junction come from the nervus spermaticus superior. In the epididymal head, small nerve bundles deviate into the septa between the ductules. Single fibres establish a dense network within the muscular coat of the ductuli. At the transition to the extratesticular rete, this network ends abruptly. Nerve fibres in the confines of the rete are associated with blood vessels or proceed to the testicular interior, but establish no relationships with the rete epithelium or the myofibroblasts of the mediastinum. The nervous network in the walls of the efferent ductules and their initial segments is not only composed of sympathetic but also parasympathetic, non-myelinated fibres. Particularly noteworthy is the abundance of calcitonin gene-related peptide (CGRP)- and substance P (SP)-containing axons around the initial segments. Both neuroproteins are consistent markers for sensory neurones. Taken together, it can be assumed that the entry of seminal fluid and spermatozoa into the efferent ductules is controlled by a regulatory nervous chain provided with afferent and efferent components.

Establishment of the urogenital junction in the male bovine embryo: an ultrastructural study.

The ultrastructure of the developing extratesticular rete testis, the efferent ductules and the establishment of the urogenital junction were studied in bovine embryos and fetuses of 41 through 95 days post conceptionem. The efferent ductules originate as a new set of secondary mesonephric tubules from the dorsal aspect of the nephric giant corpuscle and grow in the direction of the Wolffian duct. Cytological differentiation of the efferent ductules proceeds in a proximo--distal direction. At about 50-60 days, the simple columnar epithelium of the proximal portions of the efferent ductules already consists of the two typical cell types, i.e. reabsorptive principal cells with an endocytotic apparatus and a brush-border and ciliated cells. The lumen of the proximal portion is temporarily filled with intraductular blood vessels and perivascular tissue which may represent vestigial rudiments of glomeruli associated with the efferent ductules. At 50 to 60 days, the extratesticular rete still has a blastema--like appearance and consists of irregular cells with abundant glycogen. Extensions of the extratesticular rete come into contact with the efferent ductules and create the first end-to-side anastomoses with the latter. Somewhat later, the separating basal laminas vanish and invading rete cells intermingle with the epithelium of the efferent ductules, thus establishing the urogenital junction.

The nerve distribution in the testis of the cat.

The autonomous innervation of the feline testis was investigated by immunohistochemistry and a modified acetylcholinesterase technique. The nerves reach the testis mainly by two routes: (1) with testicular artery and pampiniform plexus to the cranial extremity (funicular contribution), (2) from the epididymal tail to the caudal extremity (caudal contribution). Within the tunica albuginea the funicular contribution supplies the cranial two thirds, whereas the caudal third of the tunica receives its nerves via the ligamentous connection between testis and epididymal tail. The nerve bundles accompanying the testicular artery give branches to the arterial wall and the pampiniform plexus. When reaching the cranial testicular pole the bundles separate; the majority of them pass into the centrally located mediastinum testis, another large portion enters the tunica albuginea, particularly on its epididymal side. The septula testis are innervated from both sides, that is from the mediastinum and from the tunica albuginea. In the cat, contrary to other mammals, all septula are innervated. Furthermore, nerve fibers occur regularly within the testicular lobules. Generally, the testicular nerves of the cat are unmyelinated and mainly vascular nerves, but fibers are also found within the connective tissue compartments of the testis. The vast majority of all autonomous testicular nerves are postjunctional sympathetic fibers. Terminal ramifications of cholinergic fibers are exclusively observed in the wall of medium-sized arterioles within mediastinum, septula and lobuli testis. Neuropeptide Y is the most frequent peptidergic transmitter in feline testicular vascular plexuses. The amount of calcitonin gene-related peptide-positive fibers is also remarkably high in the testis, but prefers a location within the stroma of the tunica albuginea, mediastinum and septula. In the cat, Leydig cells occur not only in intertubular locations, but also as intratunical and mediastinal Leydig cells. In all three localizations solitary nerve fibers are observed between Leydig cell groups. These fibers are generally dopamin-beta-hydroxylase- and tyrosine hydroxylase-positive, some contain calcitonin gene-related peptide and, very few, substance P.

Morphogenesis of the bovine rete testis: extratesticular rete, mesonephros and establishment of the definitive urogenital junction.

The development of the extratesticular rete, the regression of the mesonephros and the establishment of the urogenital junction between rete testis and efferent ductules were investigated in 67 bovine embryos and fetuses collected in the period from day 29 through day 250 post conception. The results were obtained by immunohistochemistry and by the study of semithin sections. At about day 30, the large mesonephros contains a peculiar Malpighian body in its cranial part, generally referred to as the mesonephric giant corpuscle, which is connected to the Wolffian duct by a series of well-developed and functioning mesonephric tubules. This set of primary mesonephric tubules, however, will not participate in the formation of the definitive urogenital junction, but will regress and soon disappear completely. The efferent ductules in the bovine are represented by another set of secondary mesonephric tubules that grow out from the dorsal aspect of the mesonephric giant corpuscle at about day 50. Transiently, the lumina of the sprouting efferent ductules are plugged by invading intraductular blood vessels, probably representing rudimentary glomeruli. The proximal portions of the newly-formed efferent ductules establish side-to-end contacts with extensions of the extratesticular rete that has bypassed the regressing giant corpuscle. At 85 days, the efferent ductules have reached the Wolffian duct and open into it. At 150 days, the channels of the extratesticular rete display a patent lumen and now form end-to-end anastomoses with the efferent ductules. The proliferating mesenchymal cells surrounding the epithelia of the efferent ductules have arranged in several concentric layers at about 85 days. These mesenchymal cells are the precursors of the periductular musculature and are reached by the first nerve fibers at about day 130.

Morphogenesis of the bovine rete testis: the intratesticular rete and its connection to the seminiferous tubules.

The development of the intragonadal rete testis and the establishment of the connection between seminiferous and straight testicular tubules was studied using ultrastructural and histochemical methods in 60 bovine embryos and fetuses ranging from day 39 through day 225 post conceptionem. The methodology included a modified acetylcholinesterase (AChE) reaction as a selective marker for pre-Sertoli cells and a modified microsomal aminopeptidase (MAP) reaction as a selective marker for the epithelia of rete testis and straight testicular tubules. Between 40 and 45 days, the rete testis is predominantly an extratesticular rete situated in the cranial peduncle of the gonadal fold and in broad contact with the pro/mesonephric giant corpuscle. During this period, the intragonadal rete enters the gonad proper from its craniodorsal pole and extends into the cranial fourth of the testis. Between 60 and 110 days the rete testis attains its definitive position, extending into the central longitudinal axis as far as to the caudal fourth of the testis. For the caudal expansion of the rete testis the preceding proliferation of the mediastinal stroma is an important prerequisite. In the 40 to 45-day-old embryo the area of the testicular cords may be divided into two zones. A narrow outer zone contains plate-like cords with a thick diameter, and a larger central zone is filled with a network of thinner cords. Only the thick outer cords transform into the permanent seminiferous tubules, whereas the thinner cords in the central zone are transitory structures that disappear between 45 and 110 days. One important function of these transitory cords is to establish a continuous system of basal laminae that allows a direct connection between the central ends of the growing seminiferous tubules and the peripheral extensions of the rete testis (future straight testicular tubules). The first true straight testicular tubules become visible between 85 and 110 days. Due to a strong proliferation of the tubulus rectus-cells the straight testicular tubules elongate continuously, and the border between the rete system and the seminiferous tubules is slowly shifted towards the testicular periphery. This shift is not restricted to the prenatal period, but proceeds until after birth. At the cytological level, the formation and elongation of the straight testicular tubules is effected by proliferating cells that advance along the continuous basal lamina into the area of the seminiferous tubules. The pre-Sertoli and germ cells in this zone of invasion are separated from each other and overgrown by the tubulus rectus-cells. Exposed to the special milieu of the straight testicular tubules, pre-Sertoli and germ cells apparently cannot survive and finally disappear.

Prespermatogenesis and spermatogoniogenesis in the bovine testis.

The bovine male germ cell population was studied over the entire period from testicular differentiation in the embryo through onset of spermatogenesis in the pubertal calf. Germ cells were identified by protein gene product 9.5 immunohistochemistry and characterized by their ultrastructure. The proliferation pattern of germ cells was studied with immunohistochemical anti-Ki 67 and anti-proliferating cell nuclear antigen reactions. Germ cells with a high proliferation rate are observed from day 50 p.c. to day 80 p.c. These cells are in transition from primordial germ cells to prespermatogonia. From day 80 p.c. until approximately the 15th postnatal week the germ cells present are identified as prespermatogonia. From day 80 p.c. to day 200 p.c. germ cell multiplication decreases continuously; then the prespermatogonia enter a phase of relative mitotical quiescence that lasts until the 4th postnatal week. Between the 4th and the 15th postnatal week, testicular tubular diameters grow from 40 to 80 microm and the prespermatogonia resume their proliferation. In seminiferous tubules with diameters between 80 and 120 microm, found in animals between 18 and 27 weeks of age, a central lumen is normally still absent. During this period germ cell proliferation reaches a second maximum. The cells involved represent the members of the spermatogonia stem and precursor cell line kinetically interpolated between the prespermatogonia and the first differentiating A-spermatogonia. This second phase of prepubertal germ cell multiplication coincides with the period when the pre-Sertoli cells transform into adult-type Sertoli cells and enter the G0-phase for the rest of life.

The significance of rudimentary nephrostomial tubules for the origin of the vertebrate gonad.

Initial gonadal development was studied in 30- to 40-day-old bovine embryos. The results were interpreted in conjunction with findings on pro- and mesonephric organization in larval forms of Ichthyophis kohtaoensis (Gymnophiona, Amphibia). In bovine embryos vestigial nephrostomial tubules are the immediate precursors of the blastemas for adrenocortical, rete, gonadal and Mullerian infundibular development. From the study of Ichthyophis it can be concluded that the vestigial nephrostomial tubules seen in the bovine embryo are pronephric and not mesonephric in nature. As a consequence, the indifferent mammalian gonad is defined as a modified homologue of the pronephros situated in the zone of pro-/mesonephric overlapping. Such an overlapping of the two kidney generations in the fully developed state is clearly seen in Ichthyophis. Overlapping of the mesonephros with the modified pronephros (gonad) is necessary to allow intercalation of mesonephric tubules (efferent ductules in mammals) into the male seminal excurrent duct system.

On the innervation of the donkey testis.

The innervation pattern of the adult donkey testis was investigated by immunohistochemistry and acetylcholinesterase histochemistry. Autonomous nerves reach the testis by three access-routes as funicular, mesorchial and caudal contributions. From these, the funicular contribution accompanying the testicular artery and pampiniform plexus is the strongest and most important one. Testicular innervation in the donkey is not uniform. The spermatic cord as well as the epididymal region, cranial and caudal poles (tunica albuginea and adjacent parenchyma and stroma) are well innervated, mostly by vascular nerves. Towards the free border of the testis, the nerve density in the tunica albuginea decreases continuously. In the interior of the gonad, approximately one third of the testis, situated between the free border and the central mediastinum, is practically devoid of any innervation. The great majority of the testicular nerves demonstrated by the present techniques are non-myelinated vascular nerves which react positive for dopamine-beta-hydroxylase and tyrosine hydroxylase, thus representing postjunctional sympathetic fibers. Many of these also contain neuropeptide Y. The testicular innervation of the donkey testis is free of cholinergic fibers. Calcitonin gene-related peptide-containing nerves are found as solitary varicose axons in the wall of blood vessels, but also in stromal connective tissue of the spermatic cord, tunica albuginea and septula testis.

On the origin and prenatal development of the bovine adrenal gland.

Decisive steps of bovine prenatal adrenal development were investigated in 46 embryos and fetuses using histological, electron microscopical, immuno-, enzyme and lectin histochemical methods. About day 30, the intermediate mesoderm between the cranial mesonephros and coelomic cavity is segmentally organized. It consists of proliferating tissue complexes that are connected to the coelomic cavity by vestigial nephrostomial tubules. This segmental organization soon disappears, however, due to longitudinal fusion of the tissue complexes into a continuous joined blastema. This blastema of intermediate mesodermal (nephric) origin becomes positive for alkaline phosphatase at about 30 days, and slightly later also for acetylcholinesterase. The most cranial portions of this common blastema represent the adrenocortical anlage, the following portions the gonadal rete blastema. A reevaluation of the comparative anatomical record revealed that a nephric origin of adrenocortical or interrenal cells is a general feature of all vertebrates and that the erroneous assumption of the lateral plate-derived coelothelium as precursor of the adrenocortical (interrenal) blastema should be definitively abandoned. The first adrenomedullary precursor cells become visible in the bovine adrenal primordium at day 35. At 50 days, both components (medullary and cortical precursors) are present as interpenetrating plates and strands between large sinusoid vessels and exhibit a strong MIB-1 activity, indicative of a high proliferation rate. About day 60 the cellular proliferation slows down in some of the adrenocortical precursor cells, and the separation into a visible cortex and medulla is initiated. From about day 80 on, the medullary tissue coalesces into a large, continuous area in the interior of the gland, surrounded by a narrow cortical glomerulo-fasciculata that becomes positive for 3beta-hydroxysteroid dehydrogenase at about day 90. Autonomous nerves penetrate the blastemal region as early as day 31. When the separation into cortex and medulla starts, the nerves are more concentrated in the latter. From 130 days on, nerve fascicles reach the interior of the organ not only from its medial side, but also from the capsule surrounding the gland. The penetrating bundles traverse the zona glomerulo-fasciculata without ramification and split off at the border to the medulla. Here, in the outer zone of the medulla, they constitute a particularly dense plexus, whereas in the central medulla a less dense innervation is observed. Up until 90 days, cells with the characteristic features of primordial germ cells are present within the confines of the adrenal gland.

Identification and temporospatial distribution of bovine primordial germ cells prior to gonadal sexual differentiation.

The temporospatial distribution of bovine primordial germ cells was studied in 34 embryos (18 to 39 days). For a reliable identification of bovine primordial germ cells in varying localizations and at different developmental stages the alkaline phosphatase reaction combined with the use of selected lectins was applied. The first potential primordial germ cells were identified in an 18-day-old trilaminar embryo in the caudal wall of the proximal yolk sac at a distance of less than 100 microm from the germ disc. These cells are alkaline phosphatase-positive. but do not yet react with lectins. From 18 through 23 days, morphogenetic folding converts the flat trilaminar disc into a cylindrical embryonic body. During this folding process primordial germ cells located in the proximal yolk sac area are incorporated into the embryo when this portion of the yolk sac becomes the hind- and mid-gut. Consequently, in 23- to 25-day-old embryos putative primordial germ cells (alkaline phosphatase- and lectin-positive) are situated predominantly in the axial body region at the level of the mesonephros. When the gonadal ridge develops in this region (about day 27) it contains a certain number of primordial germ cells present from the very beginning. Thus, the assumptions of a long-range chemoattraction of primordial germ cells by the gonadal ridge, of active immigration from an extraembryonic site. or of a passive transportation via the blood stream are not necessary to explain the initial settlement of bovine primordial germ cells in the gonadal ridge. Within the gonadal ridge (days 27-31) and later in the still sexually indifferent gonadal fold (32-39 days) the primordial germ cells are unevenly distributed. Extragonadal potential primordial germ cells (alkaline phosphatase-positive, but with reduced or no lectin staining) are regularly present in large numbers in bovine embryos with indifferent gonads. Such cells occur predominantly in the paraaortal tissue and in the liver, but also in the branchial arches. The different locations of extragonadal primordial germ cells are discussed in the light of recent evidence that germ cells and haematopoietic cells share common ancestors.

The autonomous innervation of the porcine testis in the period from birth to adulthood.

The innervation of the porcine testis was studied in 20 pigs, aged from 3 days to 2.5 years, and revealed remarkable changes in the period from birth to adulthood. Testes in piglets of 3 to 5 weeks have the most intense and most constant innervation, which reaches the gonad by three different routes: the funicular, caudal and mesorchial. Nerve fibers supply the vascular structures of the spermatic cord, the tunica albuginea, nearly all the septula testis and the mediastinum. Only exceptionally are axons in contact with Leydig cells. Nearly all the testicular nerves are positive for DBH and therefore represent postganglionic sympathetic axons. From their association with blood vessels it can be concluded that the majority of nerves are vasomotor in function. No cholinergic and myelinated fibers can be detected in the porcine testis. NPY-immunoreactive fibers are the dominating peptide-containing neuronal component. In the testes of 3- and 7-day-old piglets the degree of septal and mediastinal innervation is significantly smaller than in 3- to 5-week-old animals. In 7- to 10-week-old pigs, testicular innervation shows varying degrees of withdrawal, and the testes of adult boars are completely devoid of intrinsic nerves. Only the funicular nerves supplying the testicular artery and pampiniform plexus are preserved in the adult age group. So, the vasomotor control of intratunical, septal and mediastinal vessels and of the complete micro-circulation within the testicular parenchyma is effected without any direct nerve participation in the sexually mature boar.

A quantitative morphological study of age-related changes in the donkey testis in the period between puberty and senium.

The testis of the donkey was used as a model to study age-related changes in the period between puberty and senium. From the age of 1.5 years to the middle of sexual maturity (5 to 6 years) a number of histophysiological features, all indicative of the spermatogenetic efficiency, increase continuously. Then without a longer-lasting plateau of maximal performance these features undergo continuous retrogression. Thus, the adult testis is an organ in permanent change. During its progressive period (1.5 to 5 years) the average testicular and tubular volumes treble. The increase in tubular volume is due to an increase in tubular length (from 700 m to 1600 m per testis) and tubular diameter (from 205 microns to 250 microns). Parallel to this growth, the spermatogenetic efficiency of the seminiferous epithelium rises: the number of germ cells entering meiosis increases and the cell loss by apoptosis or exfoliation decreases. During the following regressive period of the testis (5-10 years) seminiferous epithelial height and tubular diameter are again gradually reduced to 70 microns and 205 microns, respectively. The absolute number of Sertoli cells per testis decreases continuously from puberty onwards. The tubular lamina propria thickens with advancing age and at the age of 10 years, displays long irregular projections into the seminiferous epithelium.

Autonomic innervation of the bovine testis.

The autonomic nerve supply of the bovine testis is investigated in animals of different ages by means of immunohistochemistry. Staining with antiserum to protein gene product 9.5 gives the most complete results for the study of the general innervation pattern. Autonomic nerves reach the testis by three different routes: with the blood vessels of the spermatic cord (funicular nervous contribution), by the mesorchium (mesorchial nervous contribution) and by the ligamentous bridge between epididymal tail and testis (caudal nervous contribution). The vessels of the spermatic cord are densely innervated. The large vessels of the vascular layer within the tunica albuginea display a discontinuous innervation pattern. In the interior of the testis, the caudal half of the gonad is completely free of any innervation. Slight differences in arrangement and fiber composition of testicular nerves in calves and bulls point to a reduction of the innervation with advancing age. The vast majority of bovine testicular nerves are dopamine-beta-hydroxylase-positive postganglionic sympathetic axons with vasomotor function. There is no evidence for a cholinergic innervation of the bovine testis. About half of the bovine testicular nerves are neuropeptide Y-immunoreactive. In the adult, solitary calcitonin gene-related peptide-immunoreactive fibers are the only ones independent of blood vessels. The absence of an innervation in the caudal half of the testis underlines the importance of local factors and blood-borne substances for the regulation of intratesticular blood flow in the bovine.

Immunohistochemical demonstration of nerve growth factor receptor in bovine testis.

Nerve growth factor receptor (low-affinity form) was demonstrated immunohistochemically in bovine testis by using a monoclonal mouse anti-human antibody. In the 7-month-old fetus and in the early postnatal testis, the peritubular and intertubular fibroblast-like mesenchymal cells showed a strong reaction. Following differentiation of these cells into Leydig and myoid peritubular cells, the nerve growth factor receptor was no longer expressed. However, peritubular and intertubular testicular fibroblasts/fibrocytes, which are also derived from mesenchymal precursors, remained positive. Additionally, the nerve growth factor receptor was demonstrated in postnatal prespermatogonia, A-spermatogonia, I-spermatogonia and members of the spermatogonia precursor cell line; B-spermatogonia remained negative. In A-spermatogonia and I-spermatogonia, the expression of the nerve growth factor receptor was cell-cycle-dependent and was mostly observed during G1-phase. Pre-embedding ultrahistochemistry with gold-conjugated antibody followed by silver-enhancement revealed that the nerve growth factor receptor was localized at the outer cell surface. The metal granules showed a regular distribution in positive spermatogonia. In testicular fibroblasts/fibrocytes the long narrow processes were preferentially decorated.

Postnatal development of ovine seminiferous tubules: an electron microscopical and morphometric study.

Corresponding to the increasing testicular volume and the histological appearance of the testicular parenchyma, the postnatal ontogenesis of the ovine testis can be divided into five phases. During the prebubertal period (phases 1-III), seminiferous tubules are solid and contain supporting (pre-Sertoli) cells as well as up to three types of germ cells: prespermatogonia I, II and spermatogonia precursor cells. In phase I, only prespermatogonia I are present and can usually be observed at the center of the seminiferous tubules. During phase II, prespermatogonia I migrate towards the basal lamina, divide and become prespermatogonia II. Those prespermatogonia I which are not successful in establishing contact with the tubular basal lamina degenerate. In phase III, prespermatogonia II divide and differentiate into cells which function as stem cells for spermatogenesis. Morphometric data corroborate the assumption of two types of prespermatogonia in the postnatal prepubertal ovine testis. Prespermatogonia I have nuclear volumes of about 480 microns 3 and cellular volumes of about 1200 microns3. In prespermatogonia II both volumes increase to about 920 microns3 and 1800 microns3 respectively. Adult A-spermatogonia are significantly smaller and possess an average nuclear volume of about 340 microns3 and an average cellular volume of about 800 microns3. Concomitanty with the formation of the tubular lumen in puberty (phase IV), supporting cells differentiate morphologically into typical Sertoli cells. Developmental events in the germ cell population are not yet synchronized. Adulthood (phase V) is characterized by complete spermatogenesis with all stages of the seminiferous epithelial cycle.

Immunohistochemical study of seminiferous epithelium in adult bovine testis using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen (PCNA).

The distribution pattern of proliferating cell nuclear antigen (PCNA) and Ki-67 protein was studied in adult bovine seminiferous epithelium by means of immunohistochemistry using monoclonal antibodies. Tailoring the methodological protocol for each of the two proliferation markers was a necessary prerequisite for obtaining optimal results in tubular sections and whole-mounts. A-, I- and B-spermatogonia displayed PCNA-positive nuclei, except during meta-, ana- and telophases of mitosis. PCNA-negative nuclei in the basal tubular compartment, excluding those from non-cycling Sertoli cells, belonged to the spermatogonia precursor cell line. However, only about 30%, 45% and 47% of the respective A-, I-, B-spermatogonia had positive nuclei after exposure to the MIB-1 antibody directed against the Ki-67 protein. Spermatogonia with MIB-1-negative nuclei represented cells in the G1-phase. Both antibodies reacted intensely with the nuclei of preleptotene primary spermatocytes. PCNA reactivity was also present during leptotene through pachytene. Ki-67 protein expression was absent during leptotene and zygotene but was again encountered during pachytene and meiosis I and II. Anti-PCNA/anti-protein gene product 9.5 double-labelling indicated that the transition from spermatogonia precursor cells into A1-spermatogonia is not strictly synchronized in a given tubular segment, a possible reason for the flexibility in A-spermatogonial propagation seen in bovine seminiferous tubules.

Evolution and ultrastructure of the bovine spermatogonia precursor cell line.

The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in low numbers during all the stages of the seminiferous epithelial cycle in the adult. Aggregated spermatogonia precursor cells are the dominating germ cell type in the 25-week-old and 30-week-old calf. In the adult seminiferous epithelium, they cause expansion of the basal tubular compartment as they form dense groups containing up to 15 cells. These groups are observed concomitantly with cycling A-spermatogonia and preleptotenes at the beginning of spermatocytogenesis. At the end of A-spermatogonia propagation, the aggregated spermatogonia precursor cells separate and intermingle with cycling A-spermatogonia. The spermatogonia precursor cells can later be found together with I-spermatogonia as members of an interconnected cellular network of medium-sized cells. When the I-spermatogonia divide to form the smaller B-spermatogonia, the precursor cells, which stay connected with the cycling spermatogonial population, pass through a growth phase. They can now be considered as committed spermatogonia precursor cells and are continuously being transformed into A1-spermatogonia to start a new round of spermatocytogenesis. Ultrastructurally, all members of the precursor cell line are similar. However, a number of features have been found to show a quantitative increase (endoplasmic reticulum, mitochondria) or to exhibit a rising degree of complexity (nucleolus) during the progression from basal stem cells to committed spermatogonia precursor cells.