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.

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.

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.

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.

Quantitative morphology of the ovine seminiferous epithelium.

Ultrastructural features and morphometric values of ovine Sertoli and spermatogenic cells are reported with special reference to the 6 stages of the seminiferous epithelial cycle. Seminiferous tubules occupy about 83% of the testicular parenchyma. Average tubular diameter (about 275 microns) and epithelial height (about 95 microns) do not vary significantly during the cycle. From preleptotene to late diplotene the cellular volume of primary spermatocytes increases nearly five-fold; the nuclear volume increases three-fold in the same period. Secondary spermatocytes are observed exclusively during stage 4 of the seminiferous epithelial cycle. Due to partial cell necrosis and autolytic events, ovine spermatids lose a considerable amount of their cytoplasm during acrosome and maturation phases prior to spermiation. Sertoli cells occupy between 27.6% (stage 3) and 36.6% (stage 1) of the tubular epithelium. The average volume of a Sertoli cell varies between 6380 microns 3 (stage 2) and 7195 microns 3 (stage 4), the absolute surface area between 10550 microns 2 and 12305 microns 2. The irregularly contoured Sertoli cell nucleus contains a vesicular nucleolus and occupies about 7.5% of the cell. Mitochondria (about 5%) and smooth endoplasmic reticulum are other prominent organelles, but three-quarters of the Sertoli cell are taken up by cytoplasmic matrix with a well-developed cytoskeleton. Ovine Sertoli cells contain large basal lipid droplets, but no typical lipid cycle can be observed.

The bovine tubouterine junction: general innervation pattern and distribution of adrenergic, cholinergic, and peptidergic nerve fibers.

The innervation of the bovine tubouterine junction was studied in sexually mature heifers using antisera against various neuronal markers and a modified acetylcholinesterase method. The vast majority of the nerve fibres in the bovine tubouterine junction belongs to the sympathetic nervous system; peptidergic and cholinergic fibers are restricted to characteristic locations. The endosalpinx in the adovarian portion of the terminal tubal segment is poorly innervated. The mucosa of the aduterine portion and of the tubouterine transitional region proper receives a strikingly dense innervation, which is observed mainly in combination with a strong vascularisation of specialised mucosal structures. In the endometrium, perivascular nerves accompany the ascending spiral arteries but sporadic contacts between nerve fibres and uterine glands are also observed. From the muscular coat the inner longitudinal layer of the terminal tubal segment is more richly supplied by nerve fibres than the intermediate circular and outer longitudinal layers of the tubouterine junction. No changes in the innervation pattern were seen during the different stages of the sexual cycle.

Expression of the proliferation-associated Ki-67 antigen in bovine testicular tubular cells during the seminiferous epithelial cycle, demonstrated with the MIB-1 antibody.

Ki-67 expression in the seminiferous tubule of the bovine testis was studied by immunohistochemistry during the seminiferous epithelial cycle using the monoclonal antibody MIB-1. Spermatogonial proliferation is most obvious in stages 5-7, and 8, when B-spermatogonia divide. A lower rate of spermatogonial propagation is observed preceding or during meiosis in stages 1-4. The MIB-1 antibody also gives positive results with some post-spermatogonial tubular cells. Preleptotenes passing through S-phase in stage 1 reveal positive nuclei. During prophase of meiosis I pachytenes react strongly, diplotenes react in an attenuated manner, while leptotenes and zygotenes stay negative. Secondary spermatocytes seen in stage 4 are positive as are the chromosomes during meta- and anaphase of the meiotic divisions. Post-meiotic spermatids are also decorated but stop Ki-67 expression abruptly at the end of stage 4. Sertoli cells are negative.

Ultrastructure and innervation of water buffalo (Bubalus bubalis) seminal vesicle.

The lining epithelium of secretory end pieces and central glandular duct in the seminal vesicle of the water buffalo (Bubalus bubalis) consists of columnar principal and small polymorphous basal cells. A system of intercellular and even intracellular canaliculi enlarges the secretory surface. The most prominent organelle of the columnar principal cells is the granular endoplasmic reticulum, forming large aggregates of parallel lamellae. Using antibodies against the neural cell adhesion molecule L1 and the neural marker protein gene product 9.5 (PGP 9.5), the innervation pattern of the seminal vesicle becomes evident. The muscular layer surrounding the propria contains a dense network of unmyelinated fibers. Thicker bundles traverse the muscular layer to reach the propria. Around glandular secretory tubules and below the epithelial lining of the glandular duct a tightly woven subepithelial plexus is observed which sends short penetrating branches into the basal zone of the epithelium. These intraepithelial nerves are devoid of Schwann cells and basal lamina (naked axons) and are situated within the intercellular spaces between principal and basal cells. Acetylcholinesterase histochemistry with short (1-2 h) incubation times, dopamine-beta-hydroxylase immunohistochemistry and ultrastructural study of transmitter-containing vesicles was performed. The results suggest that muscular contraction in the seminal vesicle is predominantly under the influence of the sympathetic nervous system, whereas secretory epithelial function is regulated by both sympathetic and parasympathetic fibers.

The epithelial lining of the bovine ejaculatory duct.

The bovine ejaculatory duct is lined by a pseudostratified columnar epithelium. Two cell types are present: small basal cells and columnar principal cells in different functional states. The basal cells are able to accumulate lipid material. The principal cells are observed in a less active state and in a state of either increased endocytosis and fluid uptake or active spermiophagy. Endocytotically active cells are characterized by an apical brush border and a system of microvesicles, multivesicular bodies and lysosomal dense bodies. Cells involved in phagocytosis of spermatozoa are mostly provided with a smooth apical border, an expanded Golgi apparatus, many phagocytic vacuoles and condensing phagolysosomes.

The mediastinum of the bovine testis.

The bovine testis has a central mediastinum consisting of longitudinally oriented rete channels and spacious lymph vessels, embedded in the mediastinal stroma. The latter represents a contractile-elastic unit and is composed of myofibroblasts, collagen bundles and accumulations of elastin, connecting the myofibroblasts. The dimension of the mediastinum varies in cross sections at different levels between 3.5 and 31.8 mm2. In one cross section approximately 30 rete channels and approximately 30 openings of straight testicular tubules are encountered. Nearly 25% of the area is occupied by thin-walled, valveless lymph vessels. Arterial convolutes, interpolated between straight centripetal and straight centrifugal branches of the testicular artery flank the rete on all sides. It is concluded that the pulsation within these convolutes together with the contractile-elastic stroma promotes lymph and rete content in a caudo-cranial direction. Chordae retis as described by Roosen-Runge and Holstein (1978) for the human testis are a common feature in the bovine mediastinum testis. The rete channels are lined by a simple cuboidal or columnar epithelium. Short intraepithelial crypts are present and function as epithelial reserve for dilatation and expansion of the rate. The inventory of organelles is rather inconspicuous in the rete epithelium. The apical border bears short microvilli and gives a strong reaction for alkaline phosphatase. The basal cytoplasm contains many small to medium-sized electron-dense bodies and is site of a strong acid phosphatase reaction. The rete epithelium as a whole reacts strongly with leucine aminopeptidase, the marker enzyme of the testicular excurrent duct system. Many free mononuclear cells, mostly macrophages, are observed in the basal half of the rete epithelium.

Ring-substituted [1,2-bis(4-hydroxyphenyl)ethylenediamine]dichloroplatinum (II) complexes: compounds with a selective effect on the hormone-dependent mammary carcinoma.

[1,2-Bis(4-hydroxyphenyl)ethylenediamine]dichloroplatinum (II) complexes with one substituent in the 2-position (CH3, CF3, F, Cl, Br, I: meso- and d,l-1-PtCl2, meso-(3-5)-PtCl2, meso-(7 and 8)-PtCl2) or two substituents in the 2,6-positions (CH3, Cl: meso-2-PtCl2, meso- and d,l-6-PtCl2) in both benzene rings were synthesized and tested for estrogenic and cytotoxic activities. Two complexes (meso-6-PtCl2 and meso-7-PtCl2) possess both effects. In comparative tests on estrogen receptor positive and negative mammary tumors in cell culture (MCF 7, ER+ and MDA-MB 231, ER-) and in animals (MXT, ER+ and MXT, ER-, mouse), meso-6-PtCl2 shows a selective effect on the estrogen receptor positive mammary carcinoma. A further increase of efficacy was achieved with the water-soluble (sulfato)platinum(II) derivative (meso-6-PtSO4). On the DMBA-induced hormone dependent mammary carcinoma of the SD rat, meso-6-PtSO4 is significantly more active than its ligand (meso-6) and cisplatin.

Castration cells in rat adenohypophysis after long-term alcohol consumption.

Histological and ultrastructural changes of hypophyseal gonadotropic cells of rats which received a 15% solution of ethyl alcohol for 6 months were studied. Light microscopy revealed hyperplasia and hypertrophy of these cells; some contained a vacuole of varying size. Ultrastructural analysis showed that this vacuole originated from, and anastomosed with, dilated cisternae of the granulated endoplasmic reticulum. Vacuolated cells in the pituitary of alcoholized rats were identical with cells observed after surgical or chemical castration. The development of these alcohol-induced castration cells is achieved in four stages. The first stage was characterized by beginning hydropic degeneration and other still reversible alterations. Cells in the second and third stages seemed already irreversibly on their way to decay. The fourth stage was represented by typical signet-ring cells. Our results offer a morphological basis to the adoption of a biphasic effect of alcohol on gonadotropic secretion.

Dichloro[1,2-bis(4-hydroxyphenyl)ethylenediamine]platinum(II) complexes: an approach to develop compounds with a specific effect on the hormone-dependent mammary carcinoma.

Stereoisomeric dichloro [1,2-bis(4-hydroxyphenyl)ethylenediamine]platinum(II) complexes (meso-3a, (+/-)-3b, (+)-3c, (-)-3d) and their N,N'-dibutyl derivatives (meso-4a, (+/-)-4b, (+)-4c, (-)-4d) were synthesized and tested on antitumor activity. The most active compound, 3d, shows a modest inhibition of the [3H]estradiol receptor interaction and causes a marked effect on the growth of the hormone-dependent human MCF 7 breast cancer cell line. It is also active on the hormone-independent human MDA-MB 231 breast cancer cell line, on the ADJ/PC6 plasmacytoma of the Balb/C mouse, and on the L 5222 leukemia of the BD IX rat. Apparently the inhibition of the MCF 7 cell line is not mediated by the estrogen receptor system. Histopathological studies on 3d revealed very low toxicity.

3,4-bis(3'-hydroxyphenyl)hexane--a new mammary tumor-inhibiting compound. Studies on the mechanism of action on the DMBA-induced, hormone-dependent mammary tumor of the rat.

Biochemical properties, such as the activities of eight carbohydrate-metabolism-linked enzymes and four acid hydrolases, and histological characteristics of growing and regressing DMBA-induced mammary tumors of the SD-rat after ovariectomy or treatment of the host with hexesterol, tamoxifen, and 3,4-bis(3'-hydroxyphenyl)hexane were determined. Significant differences were found between growing and regressing tumors regardless of the treatment animals had been subjected to. Only few differences in biochemical parameters could be found within the group of tumors regressing due to the applied therapy. The histological signs of regressing tumors were very diverse, but no phenomenon typical of a specific treatment could be found. It cannot be decided whether the partial antiestrogen, 3,4-bis(3'-hydroxyphenyl)hexane unfolds its antimammary tumor activity by means of its estrogenic or its antiestrogenic potency. The hypothesis that estrogens inhibit mammary tumor growth by directing neoplastic cell metabolism toward secretion is not supported by these findings.

Ultrastructural evidence for phagocytosis of spermatozoa in the bovine rete testis and testicular straight tubules.

Ultrastructural examination showed that the epithelium of the bovine rete testis and the tubuli recti could phagocytose spermatozoa. Macrophages were regularly found in the basal parts of the epithelial cells and could be involved in the removal of degraded sperm material.