Cyclic modulation of sertoli cell junctional complexes in a seasonal breeder: The mink (Mustela vison)

The development and modulation of Sertoli cell junctions was studied in newborn and adult mink during the active and inactive spermatogenic phases. The techniques used were electron microscopy of freeze-fractured replicas and thin sections of tissues infused with horseradish peroxidase as a junction permeability tracer. In the newborn, freeze-fractured developing junctions had either spherical or fibrillar particles. In addition, Junctional domains where particles were associated preferentially with the E-face, and others where particles were associated preferentially with the P-face, were found developing either singly or conjointly within a given membrane segment, thus yielding a heterogeneous Junctional segment. Coincidently with the development of a tubular lumen and the establishment of a competent blood-testis barrier, Junctional strands were composed primarily of particulate elements associated preferentially with the E-face. In adult mink during active spermatogenesis, cell junctions were found on the entire lateral Sertoli cell plasma membrane from the basal to the luminal pole of the cell. In the basal third of the Sertoli cell, membranous segments that faced a spermatogonium or a migrating spermatocyte displayed forming tight, gap, and adherens junctions. In the middle third, abutting membrane segments localized above germ cells were involved in continuous zonules and in adherens junctions. In the apical or luminal third, the zonules were discontinuous, and the association of Junctional particles with the E-face furrow was lost. Gap junctions increased in both size and numbers. Junctional vesicles that appeared as annular gap and tight-junction profiles in thin sections or as hemispheres in freeze-fracture replicas were present. Reflexive tight and gap junctions were formed through the interaction of plasma membrane segments of the same Sertoli cell. Internalized Junctional vesicles were also present in mature spermatids. During the inactive spermatogenic phase, cell junctions were localized principally in the basal third of the Sertoli cell; Junctional strands resembled those of the newborn mink. During the active spermatogenic phase, continuous zonules were competent in blocking passage of the protein tracer. During the inactive phase the blood-testis barrier was incompetent in blocking entry of the tracer into the seminiferous epithelium. It is proposed that modulation of the Sertoli cell zonules being formed at the base and dismantled at the apex of the seminiferous epithelium follows the direction of germ cell migration and opposes the apicobasal direction of junction formation reported for most epithelia. The arrest of spermatogenesis coincides with dramatic changes in the dynamic modifications of Sertoli cell zonules.     

Endogenous type C RNA virus of mink (Mustela vison).

A type C RNA virus was isolated from mink lung cell line (American Type Culture Collection No. CCL 64) which had been cocultivated with 5-bromodeoxyuridine (BUDR)-treated mouse spleen cells. The virus has type C RNA virus morphology as demonstrated by electron microscopy. The complement fixation and immunofluorescent tests performed with mouse anti-p30 antisera show a distinctive difference between mink and mouse type C viruses. Complement fixation tests also indicate that mink type C virus is antigenically different from rat, feline leukemia, feline endogenous (RD-114), baboon, and woolly monkey type C viruses. The virus propagates in cells of mouse, rat, cat, sheep, dog, and human origin, but not in bovine (MDBK) or simian (BSC-1) cells. The infection of rabbit (SIRC) cells and cells of virus origin (mink lung) was followed by delayed and low-titer polymerase release in tissue culture media. The virus sediments in sucrose density gradients as a broad band of densities, 1.13–1.17 g/ml, and contains 70 and 4S RNA. The protein profile is similar to that observed in other mammalian type C viruses. The DNA complementary to the poly(A)-containing virion RNA hybridized to a high degree (72%) with the RNA from virus-producing mink lung cells but not with the RNA from mouse cell lines or uninfected mink lung cell line. The nucleotide sequences homologous to mink viral cDNA were found in mink cell DNA from both virus-producing and nonproducing cells, but not in the DNA of mouse, rat, or feline origin. The virus here described therefore represents an endogenous mink type C virus.     

Some observations on the cerebral arterial circles of mink (Mustela vison)

With the exception of a brief allusion to an unidentified species of Mustela with regard to cerebral vascular studies by de Vriese ('05), major information concerning the circle of Willis in mink was nonexistent until the present investigation. Brains of mink in which the cerebral arterial circles were injected with latex and subsequently hardened in formalin, revealed the primary cerebral arterial anastomosis to be ring-like in form; all of the component vessels were patent and well formed, none was attenuated or string-like. Some of the more prominent findings included: (1) a predominance of asymmetric divergence of the posterior communicating arteries separating from the bifurcating basilar artery; (2) the presence of a posterior intercommunicating artery in all of the specimens; (3) the occasional doubling of the superior cerebellar and the posterior cerebral arteries; (4) deeply placed internal cerebral loops forming secondary arterial anastomoses between some penetrating vessels in the caudal region of the circle; other loops interconnected other penetrating vessels in the rostral region of the circle; (5) blood channels forming an intercarotid anastomosis traversed the pia mater; (6) the presence of a penetrating artery adjunctive to the recurrent artery of Heubner; (7) anastomoses between the internal and the external ophthalmic arteries, and between the internal and the external olfactory arteries forming collateral channels of communication between the intracranial and the extracranial circulations; (8) the occasional presence of an anterior communicating artery supplementing the commonly occurring azygos anterior cerebral artery which continued as a single vessel throughout its extent; (9) unjoined anterior cerebral arteries in one animal which was exceptional.     

Biliary parasite Pseudamphistomum truncatum (Opistorchiidae) in American mink (Mustela vison) and Eurasian otter (Lutra lutra) in Ireland

Native Eurasian otter (Lutra lutra) and introduced American mink (Mustela vison) carcasses collected throughout Ireland were screened for biliary parasites. Secondary intermediate hosts, Cyprinid fish, were also examined for Opistorchiid metacercariae. Twenty-nine mink and 24 otter gall bladders were screened for biliary parasites. A single mink and three otters were found to be infected with the digenetic trematode Pseudamphistomum truncatum. Eighty-nine percent of roach (Rutilus rutilus) from the River Shannon were infected with P. truncatum metacercariae, confirming the persistence of the parasite. This is the first record of the species in Ireland, and its recent introduction is probably related to the movement and release of Cyprinid fishes by anglers.     

Expression of the insulin-like growth factor II gene in polychlorinated biphenyl exposed female mink (Mustela vison) and their fetuses.

AIM: To study how polychlorinated biphenyls (PCBs) affect fetal growth and the expression of the insulin-like growth factor (IGF II) gene in the mink (Mustela vision). METHODS: Ten female mink were each exposed to 0.65 or 1.3 mg Clophen A50/day, respectively, during the reproductive season. The numbers and sizes of viable fetuses were recorded. The expression of the IGF II gene was studied by northern blotting using a mink specific IGF II cDNA probe. RESULTS: The number of viable fetuses decreased after PCB exposure in a dose dependent fashion. Expression of the IGF II gene in adult livers from PCB exposed animals was decreased, compared with control animals, in a dose dependent fashion. In contrast, IGF II expression in placentas and fetuses was unaltered. Furthermore, the maternal excretion of urinary cortisol increased in both exposed groups during the implantation period. CONCLUSIONS: Expression of the IGF II gene is downregulated by PCB exposure in the adult liver. There is also an indication that the regulation of the expression of this gene differs between adult and fetal life.     

Two components of the pineal organ in the mink (Mustela vison): their structural similarity to submammalian pineal complexes and calcification.

The pineal complex in the mink (Mustela vison) consists of a larger ventral and a smaller dorsal pineal. Both organs contain pinealocytes, neurons, glial cells, nerve fibers and synapses in an organization characteristic of nervous tissue. The cellular elements are arranged circularly around strait lumina. These lumina correspond to the photoreceptor spaces of submammalian pineals. A 9 + 0-type cilium marks the receptory pole of the pinealocytes which may form an inner-segment-like dendrite terminal in the pineal lumina. The cilia correspond to outer segments which form photoreceptor membrane multiplications in the pineal of submammalians and in certain insectivorous and mustelid mammals (bat, hedgehog, ferret). Axonal processes of the pinealocytes contain synaptic ribbons and terminate on intrapineal neurons of both organs. This pattern represents a neural efferentation of the pineal nervous tissue. The axonal processes of pinealocytes also form neurohormonal endings which pierce the perivascular limiting glial membrane in the ventral as well as in the dorsal pineal. The upper pineal ("epipineal") of the mink may correspond to the parapineal, frontal, or parietal organs of submammalian pineal complexes. Both pineals are encapsulated by the meningeal tissue of the brain stem. Afferent vasomotor axons of the meninges innervate smooth muscle cells of pineal arterioles. There are corpora arenacea in the pineal arachnoid and in the pineal nervous tissue, primarily in the ventral pineal. The localization of calcium ions detected around the membrane of pineal cells by pyroantimonate cytochemistry suggests membrane activity as the source of the calcium ions. The accumulation of calcium by the pinealocytes may be due to their neurosensory character. The mink is the first animal described to have both intrapineal and meningeal concrements like the human pineal.     

Pancreatitis associated with hyperlipoproteinaemia type I in mink (Mustela vison): earliest detectable changes occur in mitochondria of exocrine cells

Pancreatic tissue from young mink homozygous for a mutation in the lipoprotein lipase gene was studied by light and electron microscopy, with the aim of describing the earliest detectable changes in a process which rapidly progresses into overt pancreatitis. The mutation leads to hyperlipoproteinaemia, corresponding to hyperlipoproteinaemia type I in man. Assessment of relevant hepatic and pancreatic enzymes were included in the investigation. The earliest detectable changes consisted of widespread swelling and vacuolation of exocrine cells, arising mainly from swollen mitochondria. To a lesser extent, vesiculation of endoplasmic reticulum occurred. Mitochondria exhibited various changes, including cavitation and dilution of the matrix, with shortened and disorganized cristae displaced towards the periphery. Lamellar figures that developed within mitochondria were numerous. Acinar lumina were somewhat dilated, while plasma membranes were relatively well preserved and secretory granules seemed unchanged. Exfoliative processes progressively occurred, resulting in total necrosis of groups of parenchymal cells, while intercalated ducts were spared. The necrosis was rapidly followed by inflammatory reactions. The activity of the mitochondrial enzyme carnitine O-palmitoyltransferase, essential for the transport of fatty acids into the mitochondria, was lower in the pancreas than in the liver. The activity of the peroxisomal fatty acid beta-oxidation was high in the liver and low in the pancreas of both lipoprotein lipase-deficient and control mink. It is concluded that pancreatic lesions associated with hyperlipoproteinaemia start in exocrine cells, and are most probably the result of a metabolic disturbance, possibly a toxic effect of an excess of free fatty acids.     

Reinitiation of spermatogenesis by exogenous gonadotropins in a seasonal breeder,the woodchuck (Marmota monax), during gonadal inactivity

The present study was undertaken (1) to document structural and functional changes in the testes of seasonally breeding woodchuck during active and inactive states of spermatogenesis and (2) to evaluate the ability of exogenous gonadotropins to reinitiate spermatogenesis outside the breeding season. During seasonal gonadal inactivity, there were significant (P < 0.05) reductions in volumes of several testicular features (testis, seminiferous tubules, tubular lumen, interstitial tissue, individual Leydig cells, Leydig cell nuclei, and Leydig cell cytoplasm) as compared with gonadally active animals. The diameter of the seminiferous tubules was decreased by 26%, and Leydig cell numbers also declined in the regressed testes. These changes were accompanied by a decline in testosterone (T) levels in both plasma and testis, and reduction in epithelial height of accessory reproductive organs. A hormonal regimen was developed that would reinitiate spermatogenesis in captive, sexually quiescent woodchucks. A combination of PMSG and hCG markedly stimulated testicular growth and function and restored spermatogenesis qualitatively. Quantitatively normal spermatogenesis was restored in 2 of 6 treated males. Morphometric analyses revealed substantial increases in seminiferous tubular diameter and in the volume of seminiferous tubules, tubular lumen, total Levdig cells, and individual Leydig cells in the hormone-treated animals. These increased values corresponded to 99, 75, 68, 51, and 200%, respectively, of the values measured in naturally active woodchucks. Leydig cell numbers, however, remained unchanged and approximated only 31% of the number found in naturally active testes. Hormonal stimulation also resulted in a significant rise in serum T as well as in the total content of testicular T, and a marked increase in epithelial height in various accessory reproductive glands. The most effective hormonal protocol for stimulating spermatogenesis was treatment with 12.5 IU of PMSG twice a week for 4 weeks followed by 12.5 IU of PMSG + 25 IU of hCG twice a week for 4 weeks.     

The blood-testis barrier and its formation relative to spermatocyte maturation in the adult rat: a lanthanum tracer study

An electron-opaque substance, lanthanum, was utilized to determine when germ cells of the rat first cross the blood-testis barrier in adult spermatogenesis. Intravascularly perfused lanthanum was shown to surround all spermatogonia. Intravascularly perfused lanthanum was shown to surround all spermatogonia, preleptotene spermatocytes and early leptotene spermatocytes of Stage IX in the adult rat testis. Lanthanum was excluded from the spaces around more mature cells by newly-formed tight junctions between adjoining Sertoli processes. These processes had previously intervened between the leptotene cells and the basal lamina. The results are in close agreement with those of a previous study (Russell, '77a) which indicated that leptotene cells are the first cells of adult spermatogenesis to enter the intermediate compartment and to reside beyond a permeability barrier.     

Postnatal formation of the blood-testis barrier in the rat with special reference to the initiation of meiosis

Summary Postnatal formation of the Blood-Testis Barrier (BTB) in the rat was studied by either fixation in hypertonic fixative or employing lanthanum tracer. After 15 days of age, meiosis has reached different stages of spermatogenesis in differnt zones of the seminiferous cords. Only in those parts where germ cells are in the pachytene stage of meiosis do Sertoli cells form an effective barrier or tight compartment. Between 16 and 19 days of age, final formation of the BTB, which is to be found in the adult rat testis, occurs by zygotene and then leptotene stages successively entering the tight compartment. Thus, formation of a BTB by Sertoli cells does not occur synchronously along the length of the seminiferous cord but in accordance with the stage of meiosis of the associated germ cells.     

The blood-testis barrier in the toad (Bufo arenarum Hensel): a freeze-fracture and lanthanum tracer study

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.     

Junctional complexes of the blood-testis barrier in the Japanese quail (Coturnix Coturnix japonica)

This study investigated the developmental changes in the adherens junctions, gap junctions, as well as tight junctions forming the blood-testis barrier (BTB) in Japanese quail (Coturnix Coturnix japonica) testis. Testicular tissue from pre-pubertal, pubertal, adult, and aged Japanese quail were examined by immunohistochemistry and transmission electron microscopy (TEM). The tight junction proteins claudin-3, claudin-11, occludin and zonula occludens-1 (ZO-1), were generally localised in the cytoplasm of Sertoli cells, spermatogonia, and spermatocytes of pre-pubertal, pubertal, some adult birds. The adherens junction protein E-cadherin had a similar distribution pattern. During pre-pubertal development, the gap junction protein connexin-43 (Cx43) was only localised between Leydig cells in the testicular interstitium. However, TEM revealed the presence of gap junctions between cells of the seminiferous epithelium as early as the pre-pubertal stage. Furthermore, TEM confirmed the presence of tight and adherens junctions in the seminiferous epithelia of all age groups. The findings of this study document age-related differences in the immunolocalisation and intensity of the junctional proteins and the ultrastructure of the junctional complexes forming the BTB in quail testes. Additionally, the junctional complexes forming the BTB in the Japanese quail are well established prior to puberty. This study provides baseline information for the future evaluation of pathological changes in the BTB of avian species at different developmental stages.     

Effects of cytochalasin D on the integrity of the Sertoli cell (blood-testis) barrier

Ectoplasmic specializations (ES) containing packed actin microfilaments are associated with the numerous parallel rows of occluding junctions which form the Sertoli cell (blood-testis) barrier. To determine if ES regulate the structure of the occluding junctions and/or barrier permeability, we experimentally disrupted ES microfilaments in vivo with intratesticularly injected cytochalasin D (CD). Electron microscopic observations of seminiferous tubules from CD-treated (150–500 μM CD; 0.5–12 hr) animals indicated that ES was absent from regions where the Sertoli cell barrier is located. Seminiferous epithelial sheets from uninjected or vehicle-injected animals (1 DMSO: 1 saline) stained with NBD-phallacidin demonstrated the presence of patterned ES actin surrounding the basolateral regions of adjacent Sertoli cells. After exposure to CD, epithelial sheets exhibited increasingly patchy fluorescence indicating progressive F-actin disruption. Freeze-fracture replicas of CD-injected testes revealed numerous focal alterations in the region of occluding junctions which included disorganization of the parallel arrangement of junctional rows, the presence of free-ending rows, clustering of intramembranous particles (IMPs) between rows, reduction in the number of rows, and loss of IMPs on both the P-face and E-face. Tracer experiments, following CD exposure, were conducted to test the integrity of occluding junctions: lanthanum hydroxide, dextrose, or filipin was added, in separate experiments, to the fixative during perfusion-fixation. In another study, serum containing an antibody against adluminal germ cells was injected intratesticularly, and frozen sections were processed for immunofluorescence study. A final study consisted of simultaneous intratesticular infusions of CD and radiolabelled inulin with subsequent intraluminal and peritubular fluid sampling. In animals which were injected with CD, lanthanum was found to enter the adluminal compartment; fixative made hypertonic by addition of dextrose caused germ cells within the adluminal compartment to shrink and produce exaggerated intercellular spaces; filipin-cholesterol perturbations were present between some Sertoli cell junctional rows and on spermatid plasma membranes; and IgG was detected within the adluminal compartment of many seminiferous tubules. None of these adluminal manifestations was noted in control animals or those which received vehicle. Quantitatively, in the in vivo micropuncture experiments, significantly more radiolabelled inulin entered the lumen of seminiferous tubules from CD-treated animals than from those exposed to vehicle. Collectively, the data suggest that CD treatment alters the functional integrity of the Sertoli cell barrier and that this may be the result of disruption of microfilaments associated with the barrier.     

Changes in the ultrastructure of components of the blood-testis barrier in circulatory hypoxia

Changes in the ultrastructure of the blood-testis barrier in rats 30 and 60 min and 1 and 30 days after ligation of the testicular artery were studied by electron microscopy. The results showed that blocking of the blood flow to the testis causes rapidly progressive changes in all components of the blood-testis barrier. Micropinocytosis and destructive changes increase in the cytoplasm of the endotheliocytes of the capillaries, ending in microclasmatosis. The tunica propria of the seminiferous tubules is highly sensitive to ischemia. It becomes thickened, the nuclei and cytoplasmic organoids of its cellular components are deformed, and folding and infiltration of the basement membrane increase. Vacuolation of the cytoplasm of the sustentocytes is accompanied by destruction of the cell membrane and by separation of the sustentocytes from the tunica propria of the tubules.Department of Human Anatomy, Ivano-Frankovsk Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 8, pp. 223–226, August, 1979     

镉对大鼠血睾屏障的损伤及黄芪甲苷的保护作用

目的观察黄芪甲苷和SB203580对镉致大鼠血睾屏障破坏及相关蛋白表达改变的保护效应,探讨黄芪甲苷的保护机制。方法 21只成年雄性SD大鼠随机分为7组:单纯镉组[0.1%氯化镉腹腔内注射,1mg/(kg·d)],镉+黄芪甲苷组[镉剂量同上,同时注射黄芪甲苷,10mg/(kg·d)],镉+SB203580组[镉剂量同上,同时注射SB203580,100μg/(kg·d)],以上各组又分为连续处理5d和10d两个时间组,对照组腹腔内注射等量生理盐水。各实验和对照组动物均为3只。取睾丸做光学显微镜、电子显微镜观察以及免疫组织化学染色和Western blotting检测。结果 HE染色观察对照组支持细胞核染色较浅且不规则,镉组支持细胞内有空泡形成,镉+黄芪甲苷组与镉+SB203580组未见明显形态异常。免疫组织化学染色观察对照组闭锁小带-1蛋白(ZO-1)、紧密连接蛋白-11(claudin-11)阳性产物在生精上皮靠近基底部表达。镉组ZO-1、claudin-11阳性产物表达均显著降低(P0.05),镉+黄芪甲苷组与镉+SB203580组阳性产物表达低于对照组但明显高于镉组(P0.05)。超微结构观察对照组血睾屏障紧密连接形态完整,呈连续的电子密度较深致密线,镉组血睾屏障紧密连接及支持细胞均见不同程度破坏,镉+黄芪甲苷组与镉+SB203580组破坏程度较相应处理时间镉组为轻。Western blotting结果显示,镉组磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)水平明显增强(P0.05),镉+黄芪甲苷组与镉+SB203580组pp38MAPK水平虽高于对照组,但较镉组明显减弱(P0.05)。结论镉致大鼠血睾屏障ZO-1、claudin-11表达降低,紧密连接超微结构损伤,黄芪甲苷具有保护作用,其保护机制与抑制p38MAPK磷酸化有关。