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Department of Chemical Engineering and Technology, Panjab University, Chandigarh-160014, and *Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

(Received 10th June 1974)

The use of nitrogen mustards as chemotherapeutic agents in the treatment of certain neoplastic diseases has been well established by a large number of workers (Montgomery, 1959; Creech, Breuninger, Hankwitz, Polsky & Wilson, 1960; Ross, 1962). A large number of different compounds have been screened in various test systems in vitro and in vivo for anticancerous, antibacterial, antimicrobial and antifertility action and some useful results have been reported (see Mori, Clarkson, O'Connor Lawrence, 1962; Schmidt, Fradkin, Sullivan, & Flower, 1965; Burger, 1970). A series of substituted acetamidobenzene derivatives was screened for antifertility effects by the method of Khanna & Chaudhry (1968) to detect any antizygotic, blastocytotoxic, anti-implantation and abortifacient activity. The derivatives contained a mono- or bis-, 2-haloethyl moiety linked through a methylene group

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The levels of progesterone in the systemic plasma of eight normal and two ovariectomized cows were estimated by gas chromatography. During the last month of pregnancy, levels varied mostly in the 2·5 to 7·5 ng/ml range (0·5 to 3·0 ng/ml at calving). For six of the eight cows, there was a significant fall in level during this period; pooling of the data for all eight showed a highly significant fall.

Levels near the time of ovulation, whether or not this was associated with behavioural oestrus, were below 2 ng/ml ; they rose and fell during ovulation cycles as corpora lutea grew and regressed. Mean peak level for twenty-two cycles was 9·0 ng/ml and occurred on average 13 days after ovulation. The time of most rapid fall in progesterone level was, on average, 4 days before ovulation.

Mean levels during the first 14 days after oestrus in normal cycles were not different from those during the first 14 days after insemination in early pregnancies; they then declined in the cycles but not during the early pregnancies.

Mean progesterone levels in the plasma of ovariectomized cows up to 200 days after the operation were below 2 ng/ml; levels showed a slow but significant rise during this period.

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S. K. Gupta, A. B. Gilbert and M. A. Walker

Summary. Semi-serial (1 in 20) sections of ovaries were studied and only two types of atresia were identified—non-bursting and bursting. Smaller, non-yolky follicles (< 1 mm diameter) showed non-bursting atresia. Atresia in follicles > 1 mm diameter was invariably of the bursting type which involved the rupture of the follicular wall, and the extrusion of yolk and cellular debris through the rupture site into the stroma. However, this rupture site was small and consequently was not visible in every section but it could always be seen when the follicle was followed in semi-serial sections. The mitotic index of granulosa cells in bursting atretic follicles was much lower than that for normal follicles.

The most common criteria for distinguishing non-bursting atretic follicles were the extremely shrunken, irregularly shaped oocytes and the separation of the granulosa from the theca. In bursting atretic follicles, reliable indications were the presence in the ooplasm of some cells or cellular debris, and disorganization of the yolk and granulosa tissue. The presence of pycnotic nuclei in the granulosa cells was not a consistent feature of all atretic follicles of the hen.

Keywords: hen; follicles; atresia; histology

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M. R. Bansal, K. C. Kanwar and G. S. Gupta

Summary. Hyaluronidase from rhesus monkey testes was purified by detergent extraction, ammonium sulphate fractionation, Sephadex G-200 column chromatography and concanavalin A—Sepharose affinity chromatography. The purified hyaluronidase showed one protein band on acrylamide gel electrophoresis. Antibodies to the purified hyaluronidase were raised in rabbits and showed a single precipitin line by Ouchterlony gel diffusion. The enzyme had a molecular weight of 62 000. The K m was 0·5 mg/ml for hydrolysis of hyaluronic acid at 37°C. The optimum pH for the enzyme was 5·0 but activity was present over a broad pH range. The hyaluronidase was inhibited by HgCl2, CuSO4, FeSO4 and p-chloromercuribenzoate all at a concentration of 2 × 10−4 m. Cysteine protected the enzyme against HgCl2 inhibition.

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Amal Mukherjee, S. K. Dey, Jayasree Sen Gupta, C. S. Ramadoss and Z. Dickmann

Summary. The activities of phosphofructokinase, pyruvate kinase, citrate synthase and creatine kinase were determined in blastocysts from rabbits at 144 h post coitum and in similar blastocysts cultured for 24 h with or without oestradiol-17β (1 μg/ml). There was a significant increase in all the enzymes during the 24-h culture period but oestradiol had no effect.

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Jayasree SenGupta, P. D. Gupta, S. K. Manchanda and G. P. Talwar

Summary. By using an immunoperoxidase indirect antibody method, mouse blastocysts were found to bind specifically hCG and ovine LH but not FSH or the β unit of hCG. Brown peroxidase reaction products were present in the morula and increased with the formation of the blastocoele. The LH/hCG binding 'sites' may be related to the initiation of steroidogenesis in the embryo.

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H. Bagavant, P. Thillai-Koothan, M. G. Sharma, G. P. Talwar and S. K. Gupta

Female bonnet monkeys Macaca radiata (n = 8, four per group) were immunized with purified 55 kDa glycoprotein from porcine zona pellucida (ZP3) and ZP3 conjugated to the β subunit of human chorionic gonadotrophin (βhCG) using adjuvants permissible for human use (alum, muramyl dipeptide and sodium phthalyl derivative of lipopolysaccharide). The animals were monitored for anti-ZP3 antibody titres, biweekly progesterone concentrations, menstrual cyclicity and status of fertility. All the animals generated a good anti-ZP3 antibody response, continued to have ovulatory cycles, remained infertile in the presence of high anti-ZP3 antibody titres and showed no disturbance in cyclicity (except summer amenorrhoea). Examinations by laparoscope showed normal ovaries with developing follicles or corpora lutea on the surface. Fifty per cent of the animals conceived after a decline in antibody titres. Ovaries of animals that failed to regain fertility were examined for changes in morphology at times when anti-ZP3 antibody titres in the circulation were low and following a booster when titres were high. None of the ovaries showed any sign of inflammation or lymphocytic infiltration. Follicles at different stages of development were seen in all of the ovaries. No significant reduction in the number of follicles, except in one monkey (MRA 178), was observed. There was no increase in the numbers of atretic or degenerating follicles. The results showed that ZP3 immunization with permissible adjuvants could be used for immunocontraception without obvious ovarian changes.

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K V R Reddy, C Aranha, S M Gupta and R D Yedery

In the midst of the global epidemics of both unwanted pregnancies and sexually transmitted infections (STIs), options that provide protection are ideal. In the present study, nisin, a known antimicrobial peptide, was evaluated for safety and contraceptive potential in vitro and in vivo in the rabbit. A concentration of 400 μg nisin per ml was found to be spermicidal in vitro, and the effect was dose and time dependent. In vivo studies indicated that intravaginal application of 1 mg nisin blocked conception in rabbits. Repeated application of nisin (50 mg/animal per day) in rabbits for 14 consecutive days did not cause local inflammation or damage to the vaginal epithelium. In addition, the rate of diffusion of nisin into the blood via the vaginal mucosal epithelium, and its clearance from the circulation was found to be rapid. No treatment-related changes were observed in the reproductive performance of rabbits after cessation of treatment. Furthermore, no changes were observed in the gestation period, subsequent growth and survival of neonates in these animals. When male rats were given nisin orally for 13 consecutive weeks, no effect was observed on reproductive performance. The number of pups born, survival and growth of pups were unaltered. The affinity studies of nisin revealed that spermatozoa are more susceptible to nisin than red blood cells and vaginal epithelial cells. We suggest that nisin with spermicidal and antimicrobial properties could serve as a safe vaginal contraceptive for future therapeutic interventions in STIs.