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R. R. UNNITHAN and ARAVIND BHATIA

Summary.

Blood serum collected from female mice treated with oestrogen and progesterone was assayed for its ability to induce acrosome degeneration. The maximum activity was found in serum obtained from females treated with 1·0 μg oestrogen/day for 5 days. Lower doses did not produce any significant difference from the control group. Blood serum from pregnant or ovariectomized females treated with progesterone did not significantly depress the incidence of acrosome degeneration. The possibility that removal of the acrosome may not be a morphological concomitant of capacitation in mice has been discussed.

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R. Nowak and R. G. Rodway

Summary. Adult anoestrous ewes (N = 30) were given intravaginal Silastic implants containing melatonin or empty implants (N = 7) during mid-anoestrus (4 July). Implants were removed 16 days later (Group 1), 36 days later (Group 2) or 93 days later (Group 3). Blood samples were taken twice weekly for progesterone assay to monitor onset of ovarian activity. The percentage of ewes in each group showing early onset of ovarian cyclicity was significantly correlated with length of exposure to melatonin.

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R. R. CHAUDHURY and ASHA SETHI

Summary.

Changes in the pattern of cell division in the rat uterus with an intra-uterine silk thread suture in one horn were studied during the first 5 days of pregnancy.

There was no appreciable difference between the mitotic counts in the two horns on Days 1 to 4 of pregnancy, except that there was an increase in the luminal epithelial mitotic count on Day 4 of pregnancy in the horn with the device.

There was a marked difference in the luminal mitotic count between the two horns on Days 4 and 5 of pregnancy. While the mean mitotic count was 1·7 in the control horn on Day 5, it was 421 in the horn with the device. The diminution in the mitotic count seen on Day 5 of pregnancy in the control horn was not present in the horn with the device. It is possible that the effect of endogenous progesterone in causing diminished mitosis in the lumen was prevented by the presence of an IUD.

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R. Ravindra and R. S. Aronstam

Colchicine and taxol, which are known to influence tubulin function, were used to delineate the possible role of tubulin in signal transduction in the anterior pituitary lobe. Anterior pituitary lobes, obtained from adult male rats, were processed by discontinuous sucrose gradient centrifugation to obtain plasma membranes. The low K m GTPase activity (EC 3.6.1.) was assayed in 5 μg membrane protein using [γ-32 P]GTP at 37°C in an ATP-regenerating buffer containing 1 μmol unlabelled GTP l−1. Ten nmol l−1 each of colchicine, lumicolchicine and taxol maximally stimulated the GTPase activity by about 40% (P < 0.05). A time-course study revealed that 100 nmol colchicine l−1 stimulated the enzyme activity by 55, 74, 89 and 53% at 5, 10, 20 and 30 min, respectively (P < 0.05); lumicolchicine (100 nmol l−1) stimulated the GTPase activity by 44, 36, 11 and 55% at 5, 10, 20 and 30 min, respectively (P < 0.05). Taxol (100 nmol l−1) stimulated the enzyme activity by 39 and 25% at 20 and 30 min, respectively (P < 0.05).

Gonadotrophin-releasing hormone (GnRH) and thyrotrophin-releasing hormone (TRH) stimulated the low K m GTPase activity in a concentration-dependent manner, by up to 40–60% (P < 0.05). In the presence of 100 nmol colchicine l−1, the ability of GnRH or TRH to stimulate the GTPase activity was inhibited. For example, at 1 nmol GnRH l−1, the enzyme activity was stimulated from 124 to 176 pmol min−1 mg−1 protein; in the presence of 100 nmol colchicine l−1, activity stimulated by GnRH (1 nmol l−1) was only 157 pmol min−1 mg−1 protein (P < 0.05). At 10 nmol TRH l−1 the enzyme activity was stimulated from 124 to 174 pmol min−1 mg−1 protein; in the presence of 100 nmol colchicine l−1, activity stimulated by TRH (10 nmol l−1) was only 155 pmol min−1 mg−1 protein (P < 0.05). GnRH or TRH stimulation of the enzyme activity was not affected in the presence of lumicolchicine. In the presence of taxol, the stimulation of the GTPase activity by either GnRH or TRH was inhibited. GnRH (1 nmol l−1) stimulated the GTPase activity from 124 to 150 pmol min−1 mg−1 protein; in the presence of 100 nmol taxol l−1, activity stimulated by GnRH (1 nmol l−1) was only 128 pmol min−1 mg−1 protein (P < 0.05); 1 nmol TRH l−1 stimulated the GTPase activity from 124 to 174 pmol min−1 mg−1 protein; in the presence of 100 nmol taxol l−1, activity stimulated by TRH (1 nmol l−1) was only 157 pmol min−1 mg−1 protein (P < 0.05). These results indicate that these drugs inhibit hormone-stimulated G protein GTPase activity as a result of their interaction with tubulin or G protein(s) or both.

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R. Nowak and R. G. Rodway

Summary. Melatonin was administered intravaginally in Silastic tubing to adult and prepubertal ewes. In Exp. 1, ewe lambs (born early March) were given intravaginal melatonin implants at a mean age (±s.e.m.) of 7·5 ± 0·1 weeks (Group E, N = 10) or 19·4 ± 0·2 weeks (Group L, N = 10). The third group (Group C, N = 10) received empty implants. In Exp. 2 mature ewes were given implants on 13 May (Group E, N = 10)or 18 July (Group L, N = 10)or received empty implants (Group C, N = 10) on one of these two dates. Blood samples were taken twice weekly for progesterone assay. In Exp. 1 the mean age ( ± s.e.m.) at puberty (progesterone > 2 nmol/l for two consecutive samples) was 35·4 ± 0·8 weeks. Puberty was advanced by 5·2 weeks in Group L lambs, occurring at a mean age of 30·2 ± 0·7 weeks (P < 0·001). In Group E lambs the timing of puberty was unaltered, occurring at a mean age of 34·8 ± 0·6 weeks. Mature ewes in Group L (Exp. 2) showed increased incidence of ovarian activity (9/10 ewes cycling by 26 September) compared with the control ewes (1/10) (P < 0·001), but there was no effect in Group E ewes (3/10). The results demonstrate that continuous melatonin administration to adult and prepubertal ewes can mimic the effect of short days in terms of the reproductive response, and that the present and previous exposure to melatonin is critical in determining the response.

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R. J. Rodgers, M. R. Waterman, E. R. Simpson and R. R. Magness

Summary. The specific contents of cytochrome P-450scc and adrenodoxin in corpora lutea of late pregnant sheep were, respectively, 1/5 and 1/8 that of corpora lutea of the oestrous cycle, suggesting lower steroidogenic enzyme capacity in the former. The contents of Complex V proteins were also lower in the corpora lutea of late pregnancy. It was observed in the immunoblots of both Complex V and cytochrome P-450scc that immunoreactive bands of molecular weights lower than the native proteins were present in the samples from corpora lutea of late pregnancy, indicative of degradation of the native enzymes. It is concluded that corpora lutea of sheep during late pregnancy have a much lower enzyme capacity for steroidogenesis than do those of the oestrous cycle (mid-luteal phase) due to a reduction in the content of cytochrome P-450scc and adrenodoxin. The reduction in the levels of steroidogenic enzyme proteins appears to be unspecific and probably reflects an overall demise in mitochondrial functions.

Keywords: cholesterol side-chain cleavage cytochrome P-450; adrenodoxin; corpus luteum; pregnancy; sheep

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R. L. Ax, R. J. Collier and J. R. Lodge

Summary.

Roosters were fed 0·1 % caffeine mixed by weight into a standard ration. With continued dietary caffeine administration, the average fertility of eggs collected for 2 weeks from untreated pullets inseminated with semen from the treated males at 0,7 and 14 days after the start of treatment was 30·8, 33·5 and 3·3 %, respectively. After 14 days of treatment fertility was significantly lower (P < 0·001) than before (0 days) or 7 days after treatment. Semen output and sperm concentration were markedly reduced 17-21 days after treatment, and no semen could be collected from the roosters after they had received caffeine for 30 days. Removal of dietary caffeine resulted in resumption of semen production and a return of fertility to the control level. Testicular histology showed that spermatocyte divisions ceased and spermiogenesis was abnormal, although Leydig tissue and the response of the males to massage for semen collection was not affected. The effects on spermatogenesis and fertility were reversible after treatment for 30 days.

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N. B. VARIAN, R. R. MAURER and R. H. FOOTE

Summary.

The superovulatory response of Dutch-belted does receiving 0·10, 0·25, 0·50, 1·00, 2·00 and 4·00 mg of lh/kg of body weight following priming with twice daily injections of 0·50 mg of fsh each for 3 days was examined. The does were inseminated immediately following lh injection. Rabbits given the lowest level of lh did not ovulate, and an average of 1·0 ovulation points was obtained following the 0·25 mg level of lh. All does receiving ≥0·50 mg of lh ovulated, with an average of 33·7, 40·0, 45·1 and 35·6 ovulation points for the 0·50, 1·00, 2·00 and 4·00 mg levels, respectively (P>0·10). Some large unruptured follicles remained in all groups after they received lh.

The proportion of cleaved ova collected from fsh-primed does 26, 28 and 30 hr after lh administration was 68, 86 and 91% respectively, whereas in non-primed controls all fertilized ova appeared to have cleaved by 26 hr. This difference is attributed to the longer time required for ovulation to be completed in fsh-primed animals rather than a delay in cleavage rate following fertilization.

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LYNN R. FRASER, GILLIAN R. PATON and R. D. BARNES

Department of Infant Development, Clinical Research Centre, Harrow, Middlesex HA1 3UJ, and Department of Obstetrics and Gynaecology, University College Hospital, London

(Received 4th October 1974)

There has been considerable interest in recent months in the possibility of using fertilization in vitro and embryo transfer techniques to alleviate certain forms of human infertility.

These techniques have been used successfully in the rabbit (Chang, 1959; Fraser & Dandekar, 1973), mouse (Whittingham, 1968; Hoppe & Pitts, 1973) and rat (Toyoda & Chang, 1974). In general, relatively few embryos have been transferred and little reference to the offspring has been made other than to note phenotypic normality. Fertilization in vitro has also been described for human eggs (Edwards, Steptoe & Purdy, 1970) and more than twelve such eggs have subsequently been transplanted (Robertson, 1974), although no pregnancies have yet been reported (de Kretzer & co-authors, 1973). The reasons for failure are unknown, but the

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R. R. MAURER, HIDEO ONUMA and R. H. FOOTE

Summary.

Rabbit embryos in the two- and four-cell stage were placed in rabbit and bovine serum with and without the addition of 100 mg glucose per 100 ml serum and cultured for 97 hr. Maximum development in culture was to the expanding blastocyst stage. Embryos were transferred to recipient does after 0, 24, 36, 48, 62, 72, 88 and 97 hr in culture. Embryos transferred after 62 hr in culture readily developed into neonates, and one embryo cultured for 88 hr to the blastocyst stage developed into a full-term young. The addition of glucose significantly increased (P<0.005) the number of blastocysts produced in culture.