In Vivo and In Vitro Toxicity Profiles of Hexane Extract of Alpinia malaccensis Rhizome in Rat and Cell Line Models.

The objective of the study was to evaluate the potential toxicity of crude n-hexane extract of Alpinia malaccensis rhizome. The in vivo acute oral toxicity was evaluated by administering a single oral dose of the extract at 0, 300, or 2000 mg/kg body weight to female Wistar rats according to modified OECD Test Guideline 423. For the in vitro cytotoxicity study, A549, HepG2, 3T3, and COS-7 cell lines were exposed to different doses of A. malaccensis extract and cell viability was assessed adopting MTT assay followed by AO/EB staining, Hoechst staining, and comet assay with a view to compare the cellular and molecular mechanisms underlying the toxicity, if any. It was found that administration of 2000 mg/kg bw dose in in vivo oral acute toxicity study did not produce significant toxicity or mortality. No significant (p < 0.05) differences were observed for body weight and hematological and biochemical parameters compared to control after 14 days of treatment. No changes in behavior, body weight, hematological and biochemical parameters, and aspects of histopathology were observed when compared to the control. Thus, the possible oral lethal dose for A. malaccensis extract is above 2000 mg/kg body weight. The in vitro cytotoxicity analysis showed nontoxicity concentrations of the extract to be 2, 1.4, 30, and 1.4 µg/mL for A549, HepG2, 3T3, and COS-7 cells, respectively, where no apoptotic/necrotic cell death and DNA damage were observed. In conclusion, the extract of rhizome of A. malaccensis did not produce apparent cytotoxicity or acute oral toxicity, confirming the scope to use A. malaccensis as a safe food preservative and a natural therapeutic product after further subacute and chronic toxicity studies.

Detection of estrous biomarkers in the body exudates of Kangayam cattle (Bos indicus) from interplay of hormones and behavioral expressions.

Behavioral expressions and biochemical composition of body exudates are significantly altered in concert with the endocrine status, which are all clear indicators of physiological conditions of animals. In this study, we sought to infer about the reproductive physiological status of Kangayam cattle (Bos indicus) by analyzing behaviors, endocrine pattern, and body exudates and further to discover estrous biomarkers so as to facilitate timely artificial insemination/mating and to aid in aspects of conservation of the species. Therefore, in this study, we followed Kangayam cows through pre-estrous to post-estrous phases to correlate the endocrine dependence of biochemical constituents in urine and cervical mucus and sought to identify estrous biomarkers. Behavioral estrus was confirmed in 10 cows, from which urine samples were collected and subjected to determination of LH, FSH, estrogens, progesterone, proteins, and lipids. Furthermore, urinary fatty acids and proteins were profiled using gas chromatography and SDS-PAGE, respectively. The volatile compounds in the urine and cervical mucus were identified by gas chromatography-mass spectrometry analysis. The data revealed that LH, FSH, and estrogen levels increased significantly in estrous urine compared with nonestrous urine, whereas progesterone status was vice versa (P < 0.05). The lipid content was also significantly higher in estrous urine than in pre- and post-estrous urines (P < 0.05). There were also cyclical variations of volatiles and fatty acid profiles across phases of the estrous cycle. More acidic compounds were present in estrous urine, rendering it more acidic, than in pre- and post-estrous urines. Interestingly, oleic acid, which was present as a fatty acid in estrous and post-estrous urines, appeared to be a volatile in post-estrous urine and estrous cervical mucus. In addition, octanoic and butanoic acids were specific to both estrous urine and cervical mucus, indicating their possible candidature as estrous biomarkers. SDS-PAGE analysis showed pronounced expression of a 98 kDa protein in post-estrous urine, which in matrix-assisted laser desorption ionization-time of flight mass spectrometry was identified as albumin. Our results demonstrate multiple biomarkers in estrous urine and specific volatiles in cervical mucus that offer scope to develop viable estrus detection kits for Kangayam cows.

Assessment of urinary volatile compounds and proteins in the female goat Capra hircus: A pilot study to reveal potential indicators of oestrus.

Urine samples of female goats in pro-oestrus, oestrus and post-oestrus phases were analysed for finding oestrus-specific volatile compounds using gas chromatograph-mass spectrometry (GC-MS), and proteins using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fourteen urinary volatile were identified covering all three phases among which four compounds, 1-Tetradecanol, n-Pentadecanol, 3-Methylene tridecane and 2-Ethyl-1-dodecene, were unique to oestrus. Also, oestrus urine contained a 25 kDa protein, which was totally absent in pro-oestrus urine, and less-expressed in post-oestrus urine. This protein revealed to be complement C3 fragment. This pilot study, for the first time, reveals the difference in urinary volatile compounds and proteins in the female goat during the different phases of oestrous cycle. The four unique volatile compounds and a 25 kDa protein that appeared as oestrus-specific in this study warrant further investigation to consider them as urinary biomarkers of oestrus in goats.

Laser assisted anticancer activity of benzimidazole based metal organic nanoparticles.

Recent studies showed that the photothermal therapy can be effectively used for the targeted cancerous cells destruction. Hence, in the present study, benzimidazole based metal organic complex nanoparticles, dichloro cobalt(II) bis-benzimidazole (Co-BMZ) and dichloro copper(II) bis-benzimidazole (Cu-BMZ), were synthesized by reprecipitation method and their anti-cancer activity by means of photothermal effect has been studied. Transmission electron microscopy analysis shows that the particle size of Cu-BMZ is ∼100 nm and Co-BMZ is in the range between 100 and 400 nm. Zeta potential analysis ensures the stability of the synthesized nanoparticles. It is found that the nonlinear absorption of the nanoparticles increases with increase in laser power intensity. Phototoxicity of human lung cancer (A549) and the normal mouse embryonic fibroblast (NIH-3T3) cells was studied using a 650 nm laser. Even though both the cell lines were affected by laser irradiation, A549 cells show higher cell destruction and lower IC50 values than the normal cells. Docking studies were used to analyse the interaction site and the results showed that the Cu-BMZ molecules have higher dock score than the Co-BMZ molecules. The obtained results indicate that Cu-BMZ samples have lesser particle size, higher nonlinear absorption and higher interaction energy than the Co-BMZ samples.

Urine levels of luteinizing hormone as predictor of the period of ovulation for advantage of timed-artificial insemination in murrah buffalo (Bubalus bubalis).

Assessment of urine levels of luteinizing hormone (LH) for predicting the reproductive status of animals is in practice. The aim of this study was to predict the period of ovulation based on the urine levels of LH for timed-artificial insemination to increase the conception rate in buffaloes, which are naturally silent-oestrous animals. Level of LH in urine was assessed using ELISA, and a cut-off LH concentration for prediction of ovulation period was obtained using receiver operating characteristic analysis. Artificial insemination was performed before- and after -positive prediction of ovulation period adopting this method, and the rates of conception were assessed. Urine LH level of 105 mIU/ml (n = 14) was derived as a cut-off concentration which predicts the ovulation period. The buffaloes in the positively predicted group (day 1 or 2) inseminated via intracervical route had an increase in the conception rate (83.33%); however, the insemination in the before-positive-prediction group resulted in poor conception rates (day 0; 16.66%) compared to that of the naturally inseminated group (day 0; 75.0%). In conclusion, the urinary LH would possibly be a fairly reliable predictor of the ovulation period. The day when cut-off LH concentration is obtained may be taken as the most favourable time for artificial insemination, so as to attain a much better rate of conception in the buffalo.

Tributyltin-mediated hepatic, renal and testicular tissue damage in male Syrian hamster (Mesocricetus auratus): a study on impact of oxidative stress.

Organotin compounds are a versatile group of organometallic chemicals that are used in a variety of industrial and agricultural applications. Tributyltin (TBT), a common organotin, brings about severe spermatotoxic and organotoxic effects. However, information about the adverse effects of TBT on liver, kidney and testis is scanty. Hence, the present study was undertaken to elucidate the TBT-mediated oxidative stress-induced impairments in these organs. Administration of TBT through oral route at increasing doses of 50, 100 and 150 ppm for 65 days to male Syrian hamsters resulted in drastically decreased activities of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase and decreased mean levels of non-enzymatic antioxidants (reduced glutathione, vitamin C, and vitamin E) followed by a dramatic increase in the levels of lipid peroxidation in the liver, kidney and testis as compared to the control animals. Significantly high levels of serum urea, creatinine, uric acid and bilirubin were observed in TBT-treated hamsters. Also, TBT treatment induced drastic histopathological changes in the liver, kidney and testis combined with remarkable changes in serum levels of tissue injury marker enzymes Aspartate transaminases, Alkaline phosphatase and Alanine transaminase. These data affirm that exposure to TBT can lead to oxidative stress-induced damage to liver, kidney and testis.

Human serum albumin binding and cytotoxicity studies of surfactant-cobalt(III) complex containing 1,10-phenanthroline ligand.

The characteristics of the binding reaction of surfactant-cobalt(III) complex, cis-[Co(phen)2(C14H29NH2)]Cl2·3H2O (phen=1,10-phenanthroline, C14H29NH2=tetradecylamine) with human serum albumin (HSA) were studied by fluorescence and UV-vis absorption spectroscopy. In addition, the effect of the surfactant-cobalt(III) complex on the conformation of HSA was analysed using synchronous fluorescence spectroscopy. The experimental results showed that surfactant-cobalt(III) complex caused the fluorescence quenching of HSA through a combination of static and dynamic quenching. The number of binding sites (n) and apparent binding constant (K(a)) of surfactant-cobalt(III) complex (above and below the critical micelle concentration (cmc) were determined at various temperatures. According to the thermodynamic parameters, it is likely that hydrophobic interactions are involved in the binding process. The cancer chemotherapeutic potential of surfactant-cobalt(III) complex on ME-180 cervical cancer cell was determined using MTT assay and specific staining techniques. The complex affected the viability of the cells significantly and the cells succumbed through an apoptosis process as seen in the nuclear morphology and cytoplasmic features. In addition, single-cell electrophoresis indicated DNA damage.

Surfactant-cobalt(III) complexes: synthesis, critical micelle concentration (CMC) determination, DNA binding, antimicrobial and cytotoxicity studies.

A new class of surfactant-cobalt(III) complexes, cis-[Co(bpy)(2)(C(11)H(23)NH(2))Cl](2+) (1) and cis-[Co(phen)(2)(C(11)H(23)NH(2))Cl](2+) (2) (bpy=2,2'-bipyridyl, phen=1,10-phenanthroline), have been synthesized and characterized. The critical micelle concentration (CMC) values of these complexes in aqueous solution were obtained from conductance measurements. The specific conductivity data (at 298, 308, 318 and 328 K) served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (DeltaG(m)(0),DeltaH(m)(0)and DeltaS(m)(0)). The interaction between these complexes and calf thymus DNA in aqueous solution was investigated adopting electronic absorption spectroscopy, emission spectroscopy and viscosity measurements. Results suggest that the two complexes can bind to DNA via groove binding, van der Waals interactions and/or electrostatic interactions. The complexes showed moderate antibacterial and antifungal activities against certain selected microorganisms. The cytotoxic activity of the complexes on HBL-100 human breast cancer cells was determined adopting MTT assay and specific staining techniques, which revealed that the viability of the cells thus treated was significantly decreased and the cells succumbed to apoptosis as seen in the changes in the nuclear morphology and cytoplasmic features. Furthermore, the influence of complexes on normal cell lines from green monkey kidney was also determined and the results indicate that the effect is small on inhibition of viability.

Spermatotoxic effect of aflatoxin B1 in rat: extrusion of outer dense fibres and associated axonemal microtubule doublets of sperm flagellum.

Male Wistar rats were treated with aflatoxin B1 (AFB1). Live as well as methanol-fixed cauda epididymal spermatozoa were stained with acridine orange (AO) and ethidium bromide (EB) and observed under a fluorescence microscope. Giemsa-stained smears were observed in a bright field microscope. Unstained smears were observed with phase contrast illumination. The axoneme of more than 10% of the spermatozoa of treated rats had the outer dense fibres (ODFs), in varying numbers, and the associated axonemal microtubule doublets of the flagellum extruded either at midpiece-principal piece junction or connecting piece. This could be perceived in all light microscopic preparations, but AO-EB staining offered an advantage of the assessment of the viability as well. TEM observation of sections of the testis and cauda epididymidis also revealed ODF extrusion, as seen in the transverse sections of sperm flagella missing one or more ODFs and the associated axonemal microtubule doublets. In a few such sections, the extruded elements were seen in the cytoplasm, outside the mitochondrial sheath or peripheral sheath. Marginal to severe mitochondrial pathologies were observed in the spermatozoa and elongated spermatids, suggesting a link between AFB1-induced sperm mitochondrial pathology and extrusion of ODFs. However, the possibility that AFB1 treatment would disrupt the cytoskeletal proteins of the flagellum, resulting in the extrusion of ODFs, cannot be excluded. This sperm abnormality is reported for the first time as produced by a dietary toxin. Dietary aflatoxins, therefore, could also be contributory factors for the deterioration of the reproductive health of men.

Assembly of ovarian follicles in the caecilians Ichthyophis tricolor and Gegeneophis ramaswamii: light and transmission electron microscopic study.

Though much is known about various aspects of reproductive biology of amphibia, there is little information on the cellular and mechanistic basis of assembly of ovarian follicles in this group. This is especially true of the caecilians. Therefore, taking advantage of the abundant distribution of caecilians in the Western Ghats of India, two species of caecilians, Ichthyophis tricolor and Gegeneophis ramaswamii, were subjected to light and transmission electron microscopic analysis to trace the sequential changes during the assembly of ovarian follicles. The paired ovaries of these caecilians are elongated sac-like structures each including numerous vitellogenic follicles. The follicles are connected by a connective tissue stroma. This stroma contains nests of oogonia, primary oocytes and pregranulosa cells as spatially separated nests. During assembly of follicles the oocytes increase in size and enter the meiotic prophase when the number of nucleoli in the nucleus increases. The mitochondrial cloud or Balbiani vitelline body, initially localized at one pole of the nucleus, disperses through out the cytoplasm subsequently. Synaptonemal complexes are prominent in the pachytene stage oocytes. The pregranulosa cells migrate through the connective tissue fibrils of the stroma and arrive at the vicinity of the meiotic prophase oocytes. On contacting the oocyte, the pregranulosa cells become cuboidal in shape, wrap the diplotene stage oocyte as a discontinuous layer and increase the content of cytoplasmic organelles and inclusions. The oocytes increase in size and are arrested in diplotene when the granulosa cells become flat and form a continuous layer. Soon a perivitelline space appears between the oolemma and granulosa cells, completing the process of assembly of follicles. Thus, the events in the establishment of follicles in the caecilian ovary are described.

Light and electron microscopic observations of fabrication, release, and fate of biphasic secretion granules produced by epididymal epithelial principal cells of the fan-throated lizard Sitana ponticeriana cuvier.

The epididymis of the fan-throated lizard Sitana ponticeriana was examined with light and transmission electron microscopy to understand the cellular mechanisms of fabrication of secretion granules in epithelial principal cells, granule release into the lumen, and the fate of the dense structured granules after reaching the lumen. Principal cells of the ductus epididymis, except at the cauda, secrete electron-dense biphasic granules copiously, which decrease in abundance from the initial segment to corpus. The principal cell possesses a prominent Golgi apparatus and all versions of endoplasmic reticulum (ER), rough, smooth, and sparsely granulated. The material of the dense portion of the secretion granules, after processing at the Golgi apparatus, appears to accumulate in large ER cisternae in the supranuclear cytoplasm. It undergoes condensation when the cisternae become condensing vacuoles. Mitochondria appear to play a role in dense granule formation. The condensing vacuoles are displaced toward the apical cytoplasm when the material of the less dense portion is added to the condensing vacuoles at the Golgi area. Thus, the less dense and dense portions of the secretion granules are secreted and added to the condensing vacuoles separately. The composite granules are released into the lumen by exocytosis when the less dense portion merges with the luminal content, whereas the dense portion maintains its structured identity. The latter, initially measuring 1-2 microm in diameter, increases in size several times. It is inferred that these granules release their content gradually, resulting in the appearance of vacuoles, and suggesting that the granules have an insoluble matrix in which there is a sparingly soluble material. The substance leaching out of the granules appears to contribute to keeping the sperm quiescent and alive during storage in the male reproductive tract.

Ultrastructural evidence for secretion from the epithelium of ampulla ductus deferentis of the fan-throated lizard Sitana ponticeriana Cuvier.

Among reptiles, an ampulla ductus deferentis has been reported only in Squamata. Fairly detailed studies are available only for two species, the lizard Calotes versicolor (Fam: Agamidae) and the snake Seminatrix pygaea (Fam: Colubridae). The light microscopic study on C. versicolor revealed the ampulla to be a prominent organ, whereas the light and transmission electron microscopic study in S. pygaea revealed it to be discernable only in histological preparations. Further, the epithelium of the ductal portion of vas deferens as well as the ampulla of C. versicolor appears to contribute to the seminal plasma and can also phagocytose dead sperm, whereas in S. pygaea neither of these roles has been established. Thus, we hypothesize that there may be variations in the anatomy, histology, and the role of the vas deferens in general, and the ampulla in particular, of the squamate reptiles. In this study, the ductus deferens of the small fan-throated lizard Sitana ponticeriana (Fam: Agamidae) was subjected to light and transmission electron microscopic analysis. In this lizard the ampulla is more prominent than in C. versicolor. The epithelium of the ductal portion of vas deferens consists of principal cells (with features reflecting roles in endocytosis and phagocytosis of dead sperm), dark cells (which are absent in the epithelium of the ductal portion of vas deferens of snakes), and basal cells. The ampulla of S. ponticeriana is differentiated into storage and glandular portions. The epithelium of the storage portion is like that in the ductal portion of the vas deferens, whereas that of the glandular portion, consisting of dark and light principal cells and foamy cells, is tall and forms into smooth villous folds. All three cell types show evidence for a role in secretion, in all likelihood different from each other, for release into the lumen to contribute to seminal plasma. These cells do not provide evidence of a role in phagocytosis of dead sperm. It appears that within the Squamata, the ductal ampulla differs in structure as well as function. We suggest that the ductal ampulla of agamid lizards is a composite gland of the ampulla ductus deferentis and seminal vesicles of mammals.

Chronic chromium exposure-induced changes in testicular histoarchitecture are associated with oxidative stress: study in a non-human primate (Macaca radiata Geoffroy).

BACKGROUND: Reproductive toxicity of chromium is in dispute despite positive findings in rodents. Recently we reported epididymal toxicity of hexavalent chromium (CrVI) in bonnet monkeys and in this paper we report its testicular toxicity. METHODS: Adult monkeys (Macaca radiata) were given drinking water containing CrVI (100, 200, 400 p.p.m.) for 6 months and testes were removed for ultrastructural and biochemical analyses. RESULTS: CrVI treatment disrupted spermatogenesis, leading to accumulation of prematurely released spermatocytes, spermatids and uni- and multinucleate giant cells in the lumen of seminiferous tubules. Transmission electron microscopy revealed granulation of chromatin and vacuolation between acrosomal cap and manchette microtubules of elongated spermatids and in the Golgi area of round spermatids. Pachytene spermatocytes had fragmented chromatin and swollen mitochondria with collapsed cristae. Spermatocytes and spermatogonia in the basal compartment were unaffected. Macrophages containing phagocytosed sperm and dense inclusions in Sertoli cells were seen. Specific activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase and concentrations of the non-enzymatic antioxidants glutathione, vitamins A, C and E decreased, while concentrations of H(2)O(2) and hydroxyl radicals increased in the testis of chromium-treated monkeys. Withdrawal of chromium treatment for 6 months normalized spermatogenesis and the status of pro- and antioxidants in the testis. CONCLUSIONS: CrVI disrupts spermatogenesis by inducing free radical toxicity, and supplementation of antioxidant vitamins may be beneficial to the affected subjects.

Ameboid cells in spermatogenic cysts of caecilian testis.

Sertoli cells constitute a permanent feature of the testis lobules in caecilians irrespective of the functional state of the testis. The developing germ cells are intimately associated with the Sertoli cells, which are adherent to the basal lamina, until spermiation. There are irregularly shaped cells in the cores of the testis lobules that interact with germ cells at the face opposite to their attachment with Sertoli cells. These irregularly shaped (ameboid) cells first appear in the lumen of the cysts containing primary spermatocytes and are continually present until spermiation. We did not observe any cytoplasmic continuity between a Sertoli cell and an ameboid cell. Both light microscopic and TEM observations reveal a phagocytic role for the ameboid cells: they scavenge the residual bodies shed by spermatozoa. Organization of the ameboid cells is grossly different from that of the spermatogenic and Sertoli cells. They appear to develop from the epithelium at the juncture of the collecting ductule with the testis lobule.

Spermiogenesis in caecilians Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): analysis by light and transmission electron microscopy.

Spermiogenesis, known as spermateleosis in lower vertebrates, is the transformation of the round spermatid into a highly specialized spermatozoon with a species-specific structure. Spermateleosis and sperm morphology of two species of caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani, from the Western Ghats of Kerala, India, were studied using light and transmission electron microscopy. Spermateleosis is described in early, mid-, and late phases. During the early phase, the spermatid nucleus does not elongate, but the acrosome vesicle is Golgi-derived and its material is produced as a homogeneous substance rather than as discrete granules. In development of the acrosome, the centrioles shift in position to the lower half of the cell. The acrosomal vesicles take the full shape of the acrosome with the establishment of the perforatorium in midphase. An endonuclear canal develops and accommodates the perforatorium. The incipient flagellum is laid down when the proximal centriole attaches to the posterior side of the nucleus and the distal centriole connects to the proximal centriole, which forms the basal granule of the acrosome. The axial fiber also appears during midphase. The mitochondria shift in position to the posterior pole of the cell to commence establishment of the midphase. Late phase is characterized by nuclear condensation and elongation. Consequently, the final organization of the sperm is established with the head containing the nucleus and the acrosome. The undulating membrane separates the axoneme and axial fiber. Most of the cytoplasm is lost as residual bodies.

Microcanalization in the epididymis to overcome ductal obstruction caused by chronic exposure to chromium - a study in the mature bonnet monkey (Macaca radiata Geoffroy).

In order to apprehend the toxic effects of chromium, an occupational/environmental pollutant, on the epididymis, adult bonnet monkeys were exposed to chromium (VI) in their drinking water at concentrations of 100, 200 and 400 p.p.m. for a chronic period of 180 days. At the end of the experimental period, testicles and segments of epididymis from control and treated monkeys were subjected to light microscopic (resin-embedded semi-thin sections) and transmission electron microscopic analyses. Among the various changes undergone by the epididymal epithelium, the present paper describes the origin of two different kinds of microcanals, probably caused by ductal obstruction. The first type of microcanal, which appears to provide passage for spermatozoa to bypass the obstructed main duct, is comparable with the one already reported in carbendazim-treated efferent ductules of the rat. The second type of microcanal, which is novel, consisted of a lumen in the epithelium enclosed by four to five cells, which are either modified basal cells, principal cells or a hitherto unknown cell type. This novel type of microcanal is suggested to be a device to entrap the spermatozoa which reach the core of the epithelium and may be a mechanism to prevent extravasation of sperm so as to avoid an autoimmune response of spermatic granuloma formation. Thus, the present study has shown that chronic exposure to chromium (VI) through drinking water can produce pathological manifestations in the epididymal epithelium but the epididymis, being a versatile organ, is capable of overcoming such adverse situations through novel devices.

Stages in spermatogenesis of two species of caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): Light and electron microscopic study.

The sequential changes during spermatogenesis in the testis of two species of caecilians, Ichthyophis tricolor (Ichthyophiidae) and Uraeotyphlus cf. narayani (Uraeotyphliidae), of Western Ghats of Kerala, India, were traced using both histological techniques and transmission electron microscopy. The cell nests were assigned to stages in spermatogenesis based on the classification of van Oordt (1956, Thesis, Utrecht University). To the best of our knowledge, this is the first identification and ultrastructural description of stages in spermatogenesis in caecilians. The article illustrates not only the stages, but also the cell divisions, mitotic and meiotic, as specified. The observations indicate that, although caecilians have undergone considerable modifications in morphology and anatomy, including reproductive anatomy, in the context of a subterranean and concealed life, they appear to have conserved the typical amphibian pattern of spermatogenesis for the events of development of spermatids.

Histology and ultrastructure of male mullerian gland of Uraeotyphlus narayani (Amphibia: Gymnophiona).

This study reports the anatomy, histology, and ultrastructure of the male Mullerian gland of the caecilian Uraeotyphlus narayani, based on dissections, light microscopic histological and histochemical preparations, and transmission electron microscopic observations. The posterior end of the Mullerian duct and the urinogenital duct of this caecilian join to form a common duct before opening into the cloaca. The boundary of the entire gland has a pleuroperitoneum, followed by smooth muscle fibers and connective tissue. The Mullerian gland is composed of numerous individual tubular glands separated from each other by connective tissue. Each gland has a duct, which joins the central Mullerian duct. The ducts of the tubular glands are also surrounded by abundant connective tissue. The tubular glands differ between the column and the base in regard to the outer boundary and the epithelial organization. The basement membrane of the column is so thick that amoeboid cells may not penetrate it, whereas that around the base of the gland is thin and appears to allow migration of amoeboid cells into and out of the basal aspect of the gland. The epithelium of the column has nonciliated secretory cells with basal nuclei and ciliated nonsecretory cells with apical nuclei. In the epithelium of the base there are secretory cells, ciliated cells, and amoeboid cells. The epithelium of ducts of the tubular glands is formed of ciliated dark cells and microvillated light cells. The epithelium of the central duct is formed of ciliated dark cells also possessing microvilli, ciliated light cells also possessing microvilli, and microvillated light cells that lack cilia. It is regressed during March to June when the testis lobes are in a state of quiescence. The Mullerian gland is active in secretion during July to February when the testis is active in spermatogenesis.

Sertoli cells in the testis of caecilians, Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona): light and electron microscopic perspective.

The caecilians have evolved a unique pattern of cystic spermatogenesis in which cysts representing different stages in spermatogenesis coexist in a testis lobule. We examined unsettled issues relating to the organization of the caecilian testis lobules, including the occurrence of a fatty matrix, the possibility of both peripheral and central Sertoli cells, the origin of Sertoli cells from follicular cells, and the disengagement of older Sertoli cells to become loose central Sertoli cells. We subjected the testis of Ichthyophis tricolor (Ichthyophiidae) and Uraeotyphlus cf. narayani (Uraeotyphliidae) from the Western Ghats of Kerala, India, to light and transmission electron microscopic studies. Irrespective of the functional state of the testis, whether active or regressed, Sertoli cells constitute a permanent feature of the lobules. The tall Sertoli cells adherent to the basal lamina with basally located pleomorphic nuclei extend deeper into the lobule to meet at the core. There they provide for association of germ cells at different stages of differentiation, an aspect that has earlier been misconceived as the fatty matrix. Germ cells up to the 4-cell stage remain in the intercalating region of the Sertoli cells and they are located at the apices of the Sertoli cells from the 8-cell stage onwards. The developing germ cells are intimately associated with the Sertoli cell adherent to the basal lamina until spermiation. There are ameboid cells in the core of the lobules that appear to interact with the germ cells at the face opposite to their attachment with the Sertoli cells. Adherence of the Sertoli cells to the basal lamina is a permanent feature of the caecilian testicular lobules. The ameboid cells in the core are neither Sertoli cells nor their degeneration products.

Spermatotoxic effect of aflatoxin B(1) in the albino mouse.

With the background that the foodborne mycotoxin aflatoxin B(1) (AFB(1)) could be toxic to the male reproductive mechanism in man as well as wild and domestic animals, the present study was aimed at finding the effect of AFB(1) on sperm. The Swiss albino mouse was the test animal. AFB(1,) suspended in corn oil and ethanol (95:5, v/v), was administered intraperitoneally to 90-day-old mice at a daily dose of 50 microg/kg body weight for 7, 15, 35 and 45 days. The analysis consisted of fertility testing and counts, motility and abnormalities of the cauda epididymidal sperm, adopting light- as well as electron-microscopy. The fertility of the treated mice was reduced drastically. Sperm concentration in the epididymis and sperm motility decreased whereas sperm abnormalities increased. In particular, sperm abnormalities like two axonemes in a common cytoplasm, sticking together of heads/tails, etc., were noted. A higher percentage of cauda epididymidal spermatozoa than in the control mice retained the cytoplasmic droplet (CD) and such retention was dependent on the duration of the treatment. Spermatozoa retaining the CD were inhibited in motility. Sperm CD of AFB(1)-treated mice contained electron-dense spherical inclusions, which are hypothesized as lipid inclusions produced from the lamellae through the spherical vesicles of the CD. The results indicate disruption of the spermatogenic as well as androgenic compartments of the testis by AFB(1). The results also reflect an alteration of epididymal function towards the post-testicular sperm maturation process by AFB(1).