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M. S. Joshi

Summary. Incubation of cow oviducts flushed with 0·1 mg collagenase/ml, for 90 min helped to dislodge large numbers of ciliated and secretory cells. About 90–95% of the isolated epithelial cells were viable. The epithelial cells suspended in DMEM:F-12 + 10% serum attached to the plastic culture dish in 18–20 h after seeding. The ciliated cells which attached to the plastic dish lost their cilia after 4–5 days in culture. The attached cells, which proliferated to form a confluent monolayer 8–10 days after seeding in a 35-mm dish, could be subcultured at least 3 successive times. Some cell aggregates which did not attach to the culture dish proliferated into floating balls of cells. The ciliated cells in the unattached floating colonies maintained the ciliary movement for 9–10 days in the same culture medium. The primary cultures of the ciliated and the secretory cells maintained most of the histoarchitecture observed in intact epithelium. The secretory cells maintained their secretory activity of specific proteins in culture as indicated by immunocytology. The cultured cells contained keratin, a specific cytoskeletal component of epithelial cells.

Keywords: oviduct epithelium; cell-culture; microscopy; immunocytology; cow

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M. G. Rosenfeld and M. S. Joshi

Although the accumulation of uterine fluid during pro-oestrus in the rat has been well documented (Long & Evans, 1922; Warren, 1938), its exact role in the reproductive process is still not clearly understood. Joshi, Yaron & Lindner (1970) isolated and characterized an endopeptidase from the uterine luminal fluid of pro-oestrous rats and from immature rats treated with oestrogen. This enzyme was later found to be unique to the uterine fluid and was not found in blood (Joshi & Murray, 1974). A protein immunologically similar to the rat endopeptidase has also been demonstrated in the uterine washings of oestradiol-treated mice of 3 different strains (Joshi & Rosenfeld, 1976). The role of this enzyme is unknown, but it has been suggested that this oestrogen-dependent peptidase may play a role in the lysis of the zona pellucida and the penetration of the ovum by the spermatozoon (Joshi & Murray, 1974).

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M. S. JOSHI and I. M. MURRAY

Summary.

Endopeptidase isolated from the uterine fluid of oestrous rats was used to immunize rabbits. Immunoelectrophoresis of whole uterine fluid, purified uterine fluid peptidase and blood serum indicated that the peptidase is unique to uterine secretion and is not found in blood. The immunofluorescent protein tracing was carried out in frozen sections of the reproductive organs of immature rats, immature rats given oestrogen, mature cycling, pregnant and pseudopregnant rats. Specific immunofluorescence was observed in the luminal epithelium of the proximal portion of the oviduct of immature rats and mature rats irrespective of the stage of the cycle. The luminal epithelium and the epithelium of the endometrial glands of uteri obtained from pro-oestrous and oestrous rats showed the presence of the specific peptidase. The luminal and glandular epithelium of immature rat uterus showed the appearance of endopeptidase 40 hr after the injection of oestradiol. The presence of the uterine fluid peptidase was observed in the endometrial glands on Day 5 of pregnancy. Immunofluorescence analysis of ejaculated spermatozoa recovered from the uterus soon after mating showed accumulation of the enzyme at the head region.

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M. G. Rosenfeld and M. S. Joshi

Summary. The uterine endopeptidase of rats caused lysis of the zona pellucida of unfertilized rat and mouse eggs but not of fertilized rat and mouse eggs. Induction of cortical granule discharge of unfertilized eggs by treatment with boromycin and a guanidine derivative led to resistance of the zona pellucida to lysis by the endopeptidase. Lysis of the hamster zona pellucida occurred within 90 min, whatever the treatment and trypsin caused lysis of all zonae within 20 min. We suggest that after fertilization cortical granule discharge modifies the zona pellucida to prevent digestion by the endopeptidase.

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USHA M. JOSHI and SHANTA S. RAO

Summary.

The effect of Enovid, the well-known contraceptive agent, on the lactation performance has been studied in the mouse, the rat and the rabbit. Two dose levels were employed, one corresponding to twice the human dose, on the basis of mg/kg body weight and the other equivalent to twenty times the human dose. Growth curve of the litter was used as an index of efficacy of lactation. It was observed that, in rats, the steroid at both the dose levels caused 10 to 20% inhibition in growth rate of the young, while in rabbits there was an enhancement. The compound did not seem to have any effect on the lactation of mice. The amount of inhibition or enhancement was not related to the dose employed. The reasons for the differential response in the three species have been discussed.

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D. S. M. PRASAD, H. S. JOSHI and A. P. LABHSETWAR

Summary.

A silk suture inserted in one uterine horn significantly decreased the secretion of progesterone on Day 4 of pregnancy from the adjacent as compared to contralateral ovary. The concentration of oestradiol was similarly depressed on the IUD side. These results provide evidence for the local effect of an IUD on the ovary in the pregnant rat.

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M. S. JOSHI, A. YARON and H. R. LINDNER

Summary.

The interaction of components of the seminal plasma with the uterine luminal fluid of pro-oestrous rats was studied in vivo and in vitro. It was observed that 1 to 2 hr after mating with an intact male the uterine contents were transformed into a viscous gel, whose protein matrix was largely derived from the secretion of the coagulating glands. The bulk of this gel consisted of a high-molecular-weight (17S) protein, which was precipitated at 50 to 70% ammonium sulphate saturation. The gelation process required the presence, in small amount (< 1/10 by weight), of another protein fraction, precipitable at 30 to 50% ammonium sulphate concentrations from the coagulating gland fluid, as well as bicarbonate ions in concentrations normally found in the uterine luminal fluid of pro-oestrous rats. Both protein fractions contain covalently bound carbohydrate. The coagulable protein in the coagulating glands secretion differed in size, amino acid composition, carbohydrate content and in its behaviour towards ion exchangers from the previously described coagulinogen secreted by the seminal vesicles, whose clotting is initiated by the coagulating-gland-derived enzyme `vesiculase'.

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S. G. Joshi, K. M. Ebert and D. P. Swartz

Summary. Immunological and biochemical methods were employed to demonstrate the presence of progestagen-dependent proteins in human endometrium. Cytosols were prepared from proliferative and secretory phase endometria of cycling women, from decidua and decidua-rich tissues of women in early pregnancy and from decidua of tubal pregnancy. Antisera were raised in rabbits against the antigens of decidua of tubal pregnancy and decidua-rich tissues. Immunoelectrophoresis, Ouchterlony's immunodiffusion test and polyacrylamide gel electrophoresis using native gels revealed 2 antigenic proteins, designated antigens A and B, in secretory endometria, decidua-rich tissues, decidua, and in decidua of tubal pregnancy. However, only 1 antigenic protein was detected by SDS-gel electrophoresis: antigens A and B may therefore be two different proteins or two forms of a single protein. The antigens could not be detected in non-pregnancy sera or in term placentae. Double isotopic labelling (incubation of tissues with [3H]- and [14C]leucine) followed by protein fractionation methods were used to compare the in-vitro synthesis rates of antigens in proliferative tissues with those in decidua or secretory endometria. The rate of synthesis of antigens A and B was markedly higher in the decidua and secretory endometria than in the proliferative endometria. We conclude, therefore, that during progestagen-dependent transformation of proliferative phase endometria into secretory endometria and decidua in women, there is a selective stimulation of at least one species of pregnancy-associated protein.

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S. G. Joshi, K. M. Ebert and R. A. Smith

Summary. Antigens A and B, shown to be associated with the progestagendominated human endometrium, were partly purified and their properties studied. The antigens were recovered in the crude nuclei, the heavy particulate fraction and cytosol of decidua-rich tissue from early pregnancy. The antigens in cytosol were enriched by a combination of Concanavalin A–Sepharose chromatography and polyacrylamide gel electrophoresis. The immunological reactivity of the antigens after partial purification by Concanavalin A–Sepharose chromatography was retained after 30 min exposure to 4–85°C at pH 7·4, or after 2 h to pH 2–12 at 22°C. Trypsin, but not pepsin, RNase, DNase or neuraminidase, completely destroyed immunological reactivity of both antigens. The apparent molecular weight of both antigens determined by filtration on Sephadex G100 was 48 000. The isoelectric point of both antigens was approximately 4·9. The antigens were not immunologically related to transferrin, ceruloplasmin, alpha-1-antitrypsin, ferritin, uteroglobin, alpha-fetoprotein, human chorionic gonadotrophin, pregnancy-associated plasma proteins or pregnancy zone protein. Furthermore, the antisera to Antigens A and B did not react with the decidual cytosol of pregnant baboons or of pseudopregnant rats.

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T. N. S. UDUPA, USHA M. JOSHI, SHANTA S. RAO and D. S. PARDANANI

Infertility in certain males has been attributed to the presence of circulating autoantibodies to spermatozoa (Wilson, 1954; Rao & Sadri, 1959; Hanafiah, Epstein & Sobrero, 1972). Doubts have been expressed, however, about the clinical significance of these antibodies because they have been observed to be present in some fertile men (Fjallbrant, 1967; S. S. Rao, T. N. S. Udupa & S. S. Dikshit, unpublished observations). Various immunological methods, such as the sperm agglutination technique of Kibrick (Kibrick, Belding & Merril, 1952), microagglutination of spermatozoa (Franklin & Dukes, 1964), passive haemagglutination test (Rao & Sadri, 1959; Rangnekar & Rao, 1972), have been employed for detecting the presence of anti-sperm antibodies. It is possible that the tests used do not detect clinically signficant levels of antibodies in fertile men possessing such antibodies. Another possibility is that the spermspecific antibodies, though present in the blood, might not find access to the reproductive tract