The Histology and Histochemistry of the Circumanal Hepatoid Glands of the Dog*

Abstract— —The hepatoid circumanal glands are small at birth and continue enlarging throughout life until senility. They develop as buds from the epidermis of hair follicles of the region and in the adult surround the anus in an irregular circle, development being greater in the male. The cytoplasm of the gland cell contains non-lipid, non-proteinaceous granules and there appears to be no duct system connecting gland acini to hair follicles. It is suggested that the glands may be endocrine in type. SUMMARY The hepatoid circumanal glands of the dog are bipartite as Parks concludes, composed of a major hepatoid gland element and a minor sebaceous element. However, the rigid separation into a superficial sebaceous part and lower hepatoid circumanal gland made by Parks is not apparent, the sebaceous part when present being surrounded by the hepatoid circumanal gland. Hepatoid circumanal glands develop as buds from compound hair follicles and enlarge rapidly after birth; growth continues throughout life, and is greater in the male dog where cords of cells may extend into the subcutis for some ditsance. The hepatoid glands surround the anus in an irregular circle with little or no gap between the mucocutaneous-junction and the glands. From birth the gland cells contain non-proteinaceous non-lipid granules which stain lightly with eosin. It is suggested, however, that the hepatoid gland is an endocrine gland producing secretory granules which are passed to blood vessels which are extensive in this area, and that initially they are controlled only by the pars distalis, but after puberty, by the effect of this gland on the gonads. Possibly in females androgens produced by the adrenal cortex play a part. No communication could be demonstrated between circumanal gland and follicle. At birth large sebaceous glands are present at the mucocutaneous-junction, the sebaceous glands in the hepatoid gland zone develop at the same time as the latter, but they do not increase in size after puberty. It is suggested that the sebaceous glands produce sebum which protects the epidermis. Large coiled aporcine glands intermingle with the hepatoid glands and open into the hair follicles in the region. Résumé— —Les glandes circumanales hépatoides sont petites à la naissance et continuent à s'élargir durant toute la vie jusqu'à la veillesse. Elles se dévéloppent comme des bourgeons de l'épiderme de follicules du système pileux de la région et chez l'adulte entourent l'anus dans un cercle irrégulier, ce dévéloppement étant plus grand chez les mâles. La cytoplasme de cellules glandulaires contient des granules sans lipides ou protéines, et il paraît qu'il n'y a pas de systême de communication, unissant les acini glandulaires aux follicules pileux. Il a été suggéré que les glandes pourraient être de type endocrine. Zusammenfassung— —Die hepatoiden perianalen Drüsen sind bei der Geburt klein. Sie werden während des ganzen Lebens immer grösser bis zum Alter. Sie entwickeln sïch als Keime von de Epidermis der Haarfollikel des Gebietes; beim Erwachsenen umgeben sie den Anus in einem un-regelmässigen Kreise, die Entwicklung ist grösser beim männlichen Tier. Das Zytoplasma der Drüsenzelle enthält Körnchen, die Lipidund Proteinfrei sind. Anscheinend besteht kein Duktus-System, das die Drüsen mit den Haarfollikeln verbindet. Man nimmt an, dass die Drüsen endokrinen Charakter haben.     

Androgen receptor expression in normal, hyperplastic and neoplastic hepatoid glands in the dog

Neoplasms of the perianal glands are common in the dog, particularly in the male. The occurrence of these tumours appears to be hormone related and castration, without excision of the tumour, has sometimes resulted in regression of the tumour. The aim of this study was to investigate the expression of androgen receptors (AR) in normal, hyperplastic and neoplastic hepatoid glands in the dog. Thirty-one samples of canine hepatoid gland tissues were investigated. The lesions, classified according to WHO criteria, were comprised of 19 hyperplastic tissues, 10 benign lesions (2 hepatoid gland epithelioma and 8 hepatoid adenomas), and 19 carcinomas. Five samples from normal hepatoid glands were also investigated. The AR expression was evaluated by immunohistochemistry using a streptavidin-biotin peroxidase method. The immunoexpression was scored by two pathologists as the percentage of positive nuclei. The intensity of staining was also considered. AR expression was detected in all normal and abnormal glands. However, in hyperplastic tissues the percentage of positive nuclei was significantly higher than in normal tissue and especially in reserve basaloid cells. A similar increase in the percent of positive nuclei was also observed in hepatoid epitheliomas, while in hepatoid adenoma the percent of AR-immunolabelling was only slightly increased compared to normal tissue. In hepatoid carcinomas the percent of AR-positive cells was similar to that observed in benign tumours. The grade of differentiation of hepatoid carcinomas did not affect AR expression. These results demonstrate that increased AR expression is maintained throughout perianal gland cancer progression and that hepatoid gland carcinomas still express AR. Although further studies may be required to evaluate the hormonal background of these diseases, dogs bearing those carcinomas might benefit from castration or anti hormonal therapy.     

Expression of claudin‐5 in hepatoid gland biopsies

Claudins are integral membrane proteins involved in the structure of the tight junctions found in epithelial and endothelial cells. This study evaluated the expression of claudin‐5 in 67 hyperplastic and neoplastic lesions of canine hepatoid glands. These included normal hepatoid glands (n = 10), nodular hyperplasia (n = 10), adenomas (n = 12), epitheliomas (n = 15), differentiated carcinomas (n = 15) and anaplastic carcinomas (n = 15). There was intense lateral membrane expression of claudin‐5 on epithelial cells from normal hepatoid glands, nodular hyperplasia and adenomas, but expression was weaker in hepatoid gland epitheliomas. Basal reserve cells from normal glands, nodular hyperplasia, adenomas and epitheliomas never expressed claudin‐5. There was membrane‐bound immunoreactivity for claudin‐5 in selected areas of the epitheliomas where the cells exhibited typical hepatoid features. The weak expression of claudin‐5 molecule in epitheliomas may nevertheless lead to cellular disorientation, detachment and invasion. Claudin‐5 expression seemed to be helpful in distinguishing poorly differentiated carcinomas, differentiated carcinomas and epitheliomas of the hepatoid glands. Increased claudin‐5 expression by invasive anaplastic carcinomas may facilitate invasion and metastasis through the activation of matrix metalloproteinases.     

Immunohistochemical detection of growth hormone (GH) in canine hepatoid gland tumors

The aim of this study was to detect immunohistochemically means growth hormone (GH) in 24 hepatoid gland adenomas and 5 hepatoid gland carcinomas and to compare the difference of immunoreactivity between types of tumors. The tumors were classified according to the WHO standards. Tissue sections which were prepared from formalin-fixed, paraffin wax-embedded tissues from 25 male and 4 female dogs were carried out immunostaining using polyclonal primary anti-hGH and EnVision method. Of 24 hepatoid gland adenomas (perianal gland adenomas) 23 (95.8%) were positive. All 5 hepatoid gland carcinomas (perianal gland carcinomas) were positive. No statistically significant differences in percentage of labelled cells between malignant and benign tumors were seen. The present demonstration of GH in hepatoid gland tumors adds new data on GH in extra-pituitary tissues and hormon-dependent tumors.     

Mixed germ cell tumor in the eye of a dog.

A 3-year-old female neutered Staffordshire Bull Terrier presented with a mixed germ cell tumor involving the base of the iris and the ciliary body of the right eye. The tumor mass was composed primarily of packeted vacuolated, polygonal (hepatoid) cells and small round cells; epithelial cells lining tubuloacinar structures were a less prominent component. The hepatoid and round cells stained positively for alpha-fetoprotein and cytokeratin. The epithelial cells stained positively for cytokeratin only, and some contained cytoplasmic mucin droplets. The polygonal cells were interpreted as a hepatoid variant of yolk sac tumor, and the epithelial cells were considered a teratomatous component. Trabeculae of bone were observed within the mass and may have been metaplastic or a teratomatous element. Extragonadal germ cell tumors are rare in dogs and have previously been reported only in the suprasellar region. This is the first report of this tumor type in the eye of a nonhuman species.     

A malignant neoplasm resembling a circumanal gland adenocarcinoma in a dog.

In the neoplasm of this report a final diagnosis of a circumanal gland adenocarcinoma invading the vertebral column and canal was made based on the histologic appearance. No growths were noted in the circumanal gland region and the location of the primary neoplasm is open to debate.     

Microscopic Anatomy and Enzyme Histochemistry of the Canine Anal Canal

The microscopic anatomy of the anal canal was studied using routine histological methods and the light microscope in male and female dogs ranging from six months to 10 years of age. Precise anatomical boundaries were established for the zona columnaris , the zona intermedia , and the beginning of the zona cutanea. In an effort to clarify conflicting terminology concerning the anal canal, the term "anal sinus" ( sinus anales ) is applied to the shallow troughs located between the longitudinal mucosal ridges or columns (columnae anales) characteristic of the canine and human zona columnaris ; and the term "anal crypt" is used to represent and distinguish the terminal blind pockets of anal sinuses which extend into the lamina propria of the zona intermedia .
Anal glands were identified routinely in the z. columnaris and z. intermedia of the anal canal; dilated excretory ducts of the anal glands were frequently encountered, often filled with inflammatory cells. PAS-positive but Sudan Black-negative secretion was demonstrated in these ducts as well as within the secretory acini. Sebaceous, circumanal and apocrine sweat glands were restricted to the z. cutanea . Moderate to high alkaline phosphatase activity in the basement membrane of circumanal glands may suggest the presence of myoepithelial cells.     

Nachweis verschiedener Hydroxysteroid-Dehydrogenasen in Circumanaldrüsen adulter Hunde

Ligh-, electronmicroscopical and histochemical examinations of the circumanal glands of adult dogs suggest that these glands take part in steroid metabolism. The glands have no excretory ducts.     

Histochemical Analyses of Anti‐Microbial Substances in Canine Perianal Skin with Special Reference to Glandular Structures

Circumanal glands are prominent features of the canine perianal skin, which are often located near to the sebaceous glands and apocrine glands. As the functional relevance of circumanal glands is yet unknown, we studied the localisation of sialic acids and anti‐microbial substances (lysozyme, immunoglobulin A, lactoferrin, β‐defensin) in these glandular structures by lectin histochemistry and immunohistochemistry. The glands exhibited a number of sialic acids that were linked to α2‐6Gal/GalNAc and α2‐3Galβ1‐4GlcNAc. Additionally, lysozyme, lactoferrin and β‐defensin could be demonstrated in the three types of skin glands, whereas IgA was only detectable in the apocrine glands. The results of the study suggest the specific significance of the circumanal glands. Independent of a certain endocrine role, their products may mainly function as protective agents to preserve the integrity of the anal region, considering that sialic acids and anti‐microbial substances are important in defence mechanisms.     

Glycoconjugate expression in normal hepatoid glands and hepatoid gland adenomas and carcinomas in dogs

Although neoplastic changes in canine hepatoid perianal glands (HPG) are quite common, the biological behaviour is sometimes difficult to assess on the basis of the histopathological examination. In an attempt to detect differences in the glycoconjugate expression between normal and neoplastic HPG, and to verify their relation to the degree of neoplastic progression, normal and neoplastic HPG were studied in 13 dogs, using 11 biotinylated lectins. In HPG adenomas the majority of the cells did not stain or stained weakly after incubation with pokeweed mitogen, LCA and UEA-I. In HPG carcinoma the basal cells lost the specific binding for WGA while heterogeneous distribution of conconavalin-A staining was observed.     

Hepatocellular carcinoma with metastasis to the spleen in a Holstein cow

Hepatocellular carcinoma (HCC) with metastasis to the spleen in a Holstein cow was studied by histopathologic and immunohistochemical methods. The tumor was characterized by a pseudoglandular (acinar) pattern with an associated fibrous stroma. Individual cells often had a "hepatoid" appearance but were interspersed with scattered cells exhibiting a clear, periodic acid-Schiff (PAS)-positive cytoplasm and small eccentric nuclei. This pattern was present in nodules found in both liver and spleen. Moreover, hepatoid tumor cells were positive for alpha-fetoprotein. Immunohistochemical studies suggest that myofibroblasts were responsible for the production of fibrous septa surrounding the pseudoglandular structures of bovine HCC. In summary, our histologic and immunohistochemical findings support a diagnosis of primary HCC with splenic metastasis. Furthermore, the associated stromal response appears to be of a myofibroblast origin. The primary etiology of bovine HCC and the significance of the intralesional, PAS-positive clear cells remain undetermined.     

Expression of p63 normal canine skin and primary cutaneous glandular carcinomas

p63, a recently identified homologue of the p53 protein, is expressed consistently in basal cells of several human multilayered epithelia. In this study, expression of p63 was determined in 31 primary cutaneous glandular carcinomas, including sebaceous, perianal (hepatoid) gland, apocrine and ceruminous carcinomas, as well as their adjacent normal skin. Similar to humans, p63 is a reliable marker for basal and myoepithelial cells in canine epidermis, cutaneous appendages and malignant apocrine and ceruminous gland neoplasms. In sebaceous carcinomas, not only basal cells, but also some sebocytes, showed nuclear staining for p63. Most mature epithelial cells in perianal gland carcinomas exhibited strong p63 expression. Based on these findings, basal/myoepithelial cells could be involved in the oncogenesis of these tumours and p63 might be used as a diagnostic marker in these lesions.     

Canine Tumour Suppressor Gene p53 - Mutation in a Case of Adenoma of Circumanal Glands

Highly conserved regions of the tumour suppressor gene p53, including the typical human tumour hot spots (codons 175, 245, 248, 249, 273 and 282), were investigated in various canine neoplasms. A mutation CGG TGG (arginine tryptophan) was detected in codon 249 in an adenoma of the circumanal gland.     

Lectin histochemistry on squamous metaplasia in different epithelial tumors of dogs.

Biotinylated lectins and avidin-biotin-peroxidase complex were used to study the correlation between cellular glycoconjugates' expression and squamous maturation in normal canine skin and in various epithelial neoplasms. Normal skin tissue was obtained from five, male, random-source dogs, 5 to 7 years old. The tumors tested, selected from the files of our Department, were fifteen squamous cell carcinomas from different tissue origin, five hepatoid perianal gland adenocarcinomas with squamous metaplasia, and fourteen solid mammary carcinomas with and without histologic evidence of squamous metaplasia. Except for mammary gland carcinomas, all tumors had been surgically excised from male dogs. Intermediate filament aggregation of twelve solid mammary gland carcinomas were studied electron microscopically. The basal and the lower spinous cells in normal skin and the less differentiated cells in squamous cell carcinomas stained moderately with Griffonia simplicifolia agglutinin-I. Spinous and granular cell layers stained strongly with Phytolacca americana mitogen and Arachis hypogaea agglutinin. Both lectins stained well-differentiated cells in squamous cell carcinomas. The electron microscopic study carried out in solid carcinomas of mammary glands revealed some relationship between the presence of intracytoplasmic tonofibrils and the binding of Griffonia simplicifolia agglutinin-I and Phytolacca americana mitogen to the tumors tested. Our results suggest that the glycosylation pattern occurring during normal keratinocyte differentiation is conserved in squamous cell carcinomas and that Griffonia simplicifolia agglutinin-I and Phytolacca americana mitogen may represent useful tools in distinguishing poorly differentiated squamous cell carcinomas from other poorly differentiated mammary epithelial tumors.     

Bovine papillomaviruses, papillomas and cancer in cattle

Bovine papillomaviruses (BPV) are DNA oncogenic viruses inducing hyperplastic benign lesions of both cutaneous and mucosal epithelia in cattle. Ten (BPV 1-10) different viral genotypes have been characterised so far. BPV 1-10 are all strictly species-specific but BPV 1/2 may also infect equids inducing fibroblastic tumours. These benign lesions generally regress but may also occasionally persist, leading to a high risk of evolving into cancer, particularly in the presence of environmental carcinogenic co-factors. Among these, bracken fern is the most extensively studied. The synergism between immunosuppressants and carcinogenic principles from bracken fern and the virus has been experimentally demonstrated for both urinary bladder and alimentary canal cancer in cows whose diets were based on this plant. BPV associated tumours have veterinary and agricultural relevance in their own right, although they have also been studied as a relevant model of Human papillomavirus (HPV). Recent insights into BPV biology have paved the way to new fields of speculation on the role of these viruses in neoplastic transformation of cells other than epithelial ones. This review will briefly summarise BPV genome organization, will describe in greater detail the functions of viral oncoproteins, the interaction between the virus and co-carcinogens in tumour development; relevant aspects of immunity and vaccines will also be discussed.     

不同采精阶段对后备北极公狐的射精量、精子活力及密度的影响

采用后备北极公狐在配种期内用６个阶段的采精频率检测公狐精液的采出率、射精量、精子活力和密度。精液采出率以３月下旬和４月上旬最高,极显著地高于３月上旬（Ｐ＜００１）;射精量以３月下旬和４月上旬最高,３月上、中旬最低（Ｐ＜００５）;精子活力以４月上旬最高,极显著地高于４月下旬（Ｐ＜００１）,但４月上旬与其他阶段差异不显著（Ｐ＞００５）;精子密度以３月中旬和４月中旬最大,３月下旬最低,二者差异显著（Ｐ＜００５）。结果表明,不同的采精阶段对后备公狐的射精量、精子活力和密度均有一定的影响,尤其对后备公狐发情开始和接近结束阶段的影响更大。     

Density of Toruli Tactiles in the Japanese Monkey

Two Japanese monkeys of both sexes were examined for density of Toruli tactiles (TT) on the shaved skin stained with methylene blue solution. Each Torulus tactilis was located by the aid of a binocular loupe and marked with white lacquer. The distribution of marked TT was estimated by a mean of TT per 2cm square (2CS) of skin on the body surface. The results obtained are as follows. The mean per 2CS on the entire body surface was approximately 8.7 in the female specimen and approximately 10.0 in the male specimen. The density of distribution of TT was highest in the temporal and buccal areas, on the neck, loins and the dorsal and lateral surfaces of the arms and forearms. Density was moderate in the parieto-occipital and gluteal areas, the back, the ventral surface of the cranial half of the trunk and the lateral surface of the thigh. Density was low, to near absence in the circumanal area, on the nose, upper eyelids, cars, ischial callosities, external genitals, palms and soles.     

Lack of Hepatocarcinogenicity of Combinations of Low Doses of 2-amino-3, 8-dimethylimidazo[4,5- f ]quinoxaline and Diethylnitrosamine in Rats: Indication for the Existence of a Threshold for Genotoxic Carcinogens

The purposes of the present study were to evaluate the hepatocarcinogenicity of concurrent treatment of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and diethylnitrosamine (DEN) in rats and to determine whether no effect levels of combinations of these two different structural categories of genotoxic hepatocarcinogens exist. Two 16-week rat hepatocarcinogenesis assays were performed using a total of 790 male F344 rats. In experiment 1, we evaluated the effects of concurrent treatment of a subcarcinogenic dose of DEN on rat hepatocarcinogenesis induced by various doses of MeIQx. In experiment 2, we determined hepatocarcinogenicities of combinations of MeIQx and DEN at subcarcinogenic doses, low carcinogenic doses and high carcinogenic doses. Quantitative analyses of glutathione S-transferase placental form (GST-P)-positive foci, a preneoplastic lesion of the liver in rats, revealed that concurrent treatment with subcarcinogenic doses of DEN did not enhance MeIQx-induced rat hepatocarcinogenicity. We also found that concurrent treatment with combinations of subcarcinogenic doses of DEN and MeIQx was not hepatocarcinogenic, indicating that the combined effects of subcarcinogenic doses of DEN and MeIQx were neither additive nor synergistic. Moreover, concurrent treatment with low carcinogenic doses of these 2 carcinogens did not show additive or synergistic effects. Synergetic effects were observed only in rats coadministered high carcinogenic doses of the 2 carcinogens. These results demonstrate the existence of no effect levels of combinations of these 2 genotoxic hepatocarcinogens, and provide new evidence supporting our idea that there is a threshold, at least a practical threshold, that should be considered when evaluating the risk of genotoxic carcinogens.     

Parasites of South African wildlife. VIII. Helminth and arthropod parasites of warthogs, Phacochoerus aethiopicus, in the eastern Transvaal

Helminth and arthropod parasites were collected from 41 warthogs, Phacochoerus aethiopicus, in the Hoedspruit Nature Reserve, eastern Transvaal. This reserve consists of a military base, which is a restricted area and is surrounded by a reserve, which is open to the public. Eleven nematode species, 1 or 2 cestode species and the larvae of 2 cestode species were recovered from the animals in the reserve, and 8 nematode species and 1 or 2 cestode species were recovered from those in the military base. Oesophagostomum spp. were generally most abundant in warthogs in the reserve during the cooler months of the year, while Probstmayria vivipara also occurred in peak numbers during the cooler months, with an additional peak in October and November 1988 in warthogs in the reserve and the base, respectively. No pattern of seasonal abundance could be determined for the other helminth species. The warthogs also harboured 8 ixodid and 1 argasid tick species, 3 flea species and 1 louse species. Adult and immature Haematopinus phacochoeri were most numerous during August and September, and the largest numbers of adult Rhipicephalus simus were present from December to April.     

Estrogen receptor independent neurotoxic mechanism of bisphenol A, an environmental estrogen

Bisphenol A (BPA), a ubiquitous environmental contaminant, has been shown to cause developmental toxicity and carcinogenic effects. BPA may have physiological activity through estrogen receptor (ER) -α and -β, which are expressed in the central nervous system. We previously found that exposure of BPA to immature mice resulted in behavioral alternation, suggesting that overexposure of BPA could be neurotoxic. In this study, we further investigated the molecular neurotoxic mechanisms of BPA. BPA increased vulnerability (decrease of cell viability and differentiation, and increase of apoptotic cell death) of undifferentiated PC12 cells and cortical neuronal cells isolated from gestation 18 day rat embryos in a concentration-dependent manner (more than 50 µM). The ER antagonists, ICI 182,780, and tamoxifen, did not block these effects. The cell vulnerability against BPA was not significantly different in the PC12 cells overexpressing ER-α and ER-β compared with PC12 cells expressing vector alone. In addition, there was no difference observed between BPA and 17-β estradiol, a well-known agonist of ER receptor in the induction of neurotoxic responses. Further study of the mechanism showed that BPA significantly activated extracellular signal-regulated kinase (ERK) but inhibited anti-apoptotic nuclear factor kappa B (NF-κB) activation. In addition, ERK-specific inhibitor, PD 98,059, reversed BPA-induced cell death and restored NF-κB activity. This study demonstrated that exposure to BPA can cause neuronal cell death which may eventually be related with behavioral alternation in vivo. However, this neurotoxic effect may not be directly mediated through an ER receptor, as an ERK/NF-κB pathway may be more closely involved in BPA-induced neuronal toxicity.