Distribution of lactate dehydrogenase in healthy and degenerative canine stifle joint cartilage.

In dogs, degenerative joint diseases (DJD) have been shown to be associated with increased lactate dehydrogenase (LDH) activity in the synovial fluid. The goal of this study was to examine healthy and degenerative stifle joints in order to clarify the origin of LDH in synovial fluid. In order to assess the distribution of LDH, cartilage samples from healthy and degenerative knee joints were investigated by means of light and transmission electron microscopy in conjunction with immunolabeling and enzyme cytochemistry. Morphological analysis confirmed DJD. All techniques used corroborated the presence of LDH in chondrocytes and in the interterritorial matrix of healthy and degenerative stifle joints. Although enzymatic activity of LDH was clearly demonstrated in the territorial matrix by means of the tetrazolium-formazan reaction, immunolabeling for LDH was missing in this region. With respect to the distribution of LDH in the interterritorial matrix, a striking decrease from superficial to deeper layers was present in healthy dogs but was missing in affected joints. These results support the contention that LDH in synovial fluid of degenerative joints originates from cartilage. Therefore, we suggest that (1) LDH is transferred from chondrocytes to ECM in both healthy dogs and dogs with degenerative joint disease and that (2) in degenerative joints, LDH is released from chondrocytes and the ECM into synovial fluid through abrasion of cartilage as well as through enhanced diffusion as a result of increased water content and degradation of collagen.

Blood-tissue barriers in the male reproductive tract of the dog: a morphological study using lanthanum nitrate as an electron-opaque tracer.

Blood-tissue barriers preventing an uncontrolled exchange of larger molecules between adjacent but metabolically separate compartments have been demonstrated in various organs. One prominent example is the blood-testis barrier which has been investigated in a number of species. A key function of this barrier is to shield developing germ cells from the immune system in order to avoid autoimmune reactions. This requirement also applies to the male excurrent duct system. Yet, very few investigations have addressed the morphology of the blood-epididymal barrier. The goal of the present study, therefore, was to revisit the blood-testis barrier in the dog and to identify the structures constituting the blood-epididymal barrier in this species. Lanthanum nitrate was used as a tracer for electron microscopy. In the testis, lanthanum had free access to the intercellular space of the seminiferous epithelium up to the Sertoli cell junctions. Similarly, epithelial tight junctions were found to represent the permeability barrier in the epididymis. The present study highlights species differences with respect to the blood-testis barrier and extends the knowledge of the blood-epididymal barrier by providing morphofunctional data in this domestic species.

Density and distribution of anionic sites on boar ejaculated and epididymal spermatozoa.

Current knowledge implies that spermatozoa successively acquire negative surface charges as they migrate through the epididymis. Until recently, however, techniques used were not amenable to statistical analysis. In the present study, a novel approach allowing numerical assessment of negative charge labelling was used in order to determine the density and distribution of anionic sites on ejaculated and maturing spermatozoa collected from six regions of the boar epididymis. Labelling was assessed quantitatively for the three morphologically distinct membrane domains on the sperm head. Statistical analysis revealed that labelling density was highest on efferent duct spermatozoa, declined up to the proximal corpus and then increased again. Densities of anionic sites on distal corpus, proximal cauda and ejaculated sperm cells were similar but significantly below the values obtained for efferent duct spermatozoa. All three sperm membrane domains underwent parallel changes. However, the overall density of negative charges on the postacrosomal segment was significantly higher as compared to the acrosomal plasma membrane. These alterations reflect sperm surface modifications through removal and addition of anionic groups. Since charge interactions are considered to play a pivotal role in sperm-egg interactions, these processes should be viewed as an integral part of sperm maturation.

Demonstration and cytochemical analysis of anionic sites on ejaculated boar spermatozoa: a scanning electron microscopy study using cationised colloidal gold.

The plasmalemma of spermatozoa bears negative charges as is the case for most mammalian cells. This has been concluded from the sperm cell's electrophoretic behaviour and from labelling experiments with various cationic probes followed by transmission electron microscopy of ultrathin sections. An overall view of the cell surface, however, is necessary in order to assess the distribution and density of the anionic sites adequately. We, therefore, used scanning electron microscopy in combination with cationised colloidal gold labelling to analyse the presence of anionic sites on ejaculated boar spermatozoa. Incubations were performed at pH 3.5, 2.5 and 1.0. Labelling was specific and bound gold particles were unequivocally identified using the backscattered electron signal. The chemical nature of the anionic sites involved was investigated by treating spermatozoa with pronase, phosphatase and neuraminidase as well as by methylation, acid hydrolysis and beta-elimination prior to cationised gold labelling. Our results suggest that besides phosphates, carboxyl groups are predominantly accountable for the binding of cationised colloidal gold. Presumptive macromolecules bearing these anionic sites are phospholipids and sialic acid residues. The combination of methods presented herewith should be of value in order to elucidate charge interactions which have been shown to play a role in cellular recognition events and adhesion.