Experiments were conducted to examine whether seasonal breeding patterns of male sheep are abrogated by thyroidectomy. In Expt 1, Welsh Mountain rams were thyroidectomized in early autumn (September) and then maintained on either 16 h light:8 h dark (long days; n = 6) or 8 h light:16 h dark (short days; n = 6) for 8 months. Intact rams (n = 6 per group) were also housed in long or short days, or in natural photoperiods. Results were similar in animals housed on long or short days. In thyroidectomized rams, plasma FSH concentrations and scrotal circumference were maintained at values typical of the breeding season throughout the investigation, whereas in intact animals both reached a nadir in December and January. In Expt 2, a further 11 rams were thyroidectomized in March and, together with 23 intact animals, were maintained thereafter in natural photoperiods. In control rams, scrotal circumference increased slowly between May and September, whereas in thyroidectomized animals the circumference increased rapidly in the first 4 weeks following thyroidectomy (3.7 ± 0.7 cm), with a further increase (5.9 ± 1.0 cm) in the next 4 weeks. The scrotal circumference of thyroidectomized rams was therefore significantly (P < 0.01) greater than that in intact animals between April and August. Plasma FSH concentrations were significantly (P< 0.01) higher in thyroidectomized than in control rams by two weeks after surgery. These results indicate that thyroidectomy overcomes the seasonal (or photorefractory) inhibition of reproductive activity in rams and supports a key role for thyroid hormones in the expression of seasonal patterns of breeding activity.
T. J. Parkinson and B. K. Follett
T. J. Parkinson and G. E. Lamming
Summary. Plasma progesterone and LH secretion patterns were examined in 18 mature dairy cows during the oestrous cycle and after insemination. Blood samples were collected every 15 min for 8 h per day on Days 3, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20 and 21 of the oestrous cycle, then, in the same cows, at the same times during early pregnancy. PGF-2α secretion rates (as determined by plasma PGFM concentrations) were also monitored on Days 14, 16 and the day of, or equivalent to, luteal regression. Mean daily plasma progesterone concentrations were similar until Day 16 in cyclic and pregnant cows, after which values in non-pregnant animals declined. Regression analysis indicated that progesterone concentrations were best described by a quadratic expression with fitted maximum values on Day 13 in non-pregnant animals but values increased linearly over the whole period to Day 21 in pregnant cows. The frequency, amplitude and area under the curve of LH episodes showed no significant differences between cyclic and pregnant animals. In pregnant cows, the amplitude and area under the curve of progesterone episodes increased linearly between Days 8 and 21, although no such increase occurred in cyclic cows. Low-level PGFM episodes were present in cyclic and pregnant cows on Days 14 and 16 after oestrus, and high amplitude episodes occurred in non-pregnant cows during luteal regression. Pregnant cows showed a significant depression of the amplitude, but not the frequency of episodes at the expected time of luteal regression. These results confirm that the corpus luteum of pregnancy secretes an increasing amount of progesterone per se and per unit of LH until at least Day 21 after mating. They further suggest that the corpus luteum of the cyclic cow may experience small episodes of PGF-2α and be subjected to initial degenerative changes by Day 14 after oestrus, some time before the onset of definitive luteolysis.
Keywords: early pregnancy; cow; LH/progesterone relationships
T. J. Parkinson, J. A. Douthwaite, and B. K. Follett
Thyroidectomy of seasonally breeding birds and mammals prevents the return to a state of sexual quiescence at the end of the breeding season. In starlings, thyroidectomy also causes premature sexual maturity. In this study, the effect of thyroidectomy upon the time of sexual maturity of prepubertal (8 week-old) ram lambs was examined. Thyroidectomy of four prepubertal and six mature rams was performed early in the spring. These and sham-operated controls were maintained in ambient photoperiods (south-west England). Scrotal circumference and serum LH, FSH, prolactin and thyroxine were measured every 2 weeks. In both the prepubertal lambs and the mature rams, scrotal circumference increased significantly within 5 weeks of thyroidectomy. FSH concentrations increased significantly in the mature rams after thyroidectomy. The relatively high FSH concentrations of thyroidectomized animals at the start of the experiment were maintained, but the FSH concentrations of intact lambs decreased during the late spring. These results provide the first indication that the timing of puberty in seasonally breeding mammals is a thyroid-dependent phenomenon.
T. J. Parkinson, L. J. Jenner, and G. E. Lamming
Summary. The secretion of prostaglandin (PG) F-2α in response to intravenous injection of 100 i.u. oxytocin on Day 18 after oestrus was determined by measuring jugular venous concentrations of 13,14-dihydro-15-keto PGF-2α (PGFM) in 7 pregnant, 6 cyclic and 2 inseminated non-pregnant heifers. Two other heifers received i.v. saline (controls). The immediate responses of pregnant heifers were smaller than in non-pregnant animals (P < 0·05), as were baseline concentrations in the post-response period (P < 0·05). Endometrial oxytocin receptor concentrations were higher in non-pregnant than pregnant heifers (P < 0·05), but PGFM response to oxytocin challenge was not correlated with oxytocin receptor concentration. Oxytocin receptor concentrations on Day 18 were positively correlated with those of plasma oestradiol on Day 17 (P < 0·01) and inversely with plasma progesterone concentrations on Day 18 (P < 0·01). These findings confirm that PGF-2α secretion in response to oxytocin challenge is attenuated in pregnant animals on the 18th day after oestrus and that, while the prevailing steroid environment is of importance in inducing oxytocin receptor activity, the secretion of PGF-2α is not subsequently limited by oxytocin receptor numbers.
The quantities of PGE-2, PGFM and PGF-2α recovered in uterine flushings taken from heifers on Day 18 were greater in pregnant than other animals (P < 0·01, P < 0·05, P < 0·001), respectively). Intrauterine concentrations of PGF-2α and PGFM were not correlated with the plasma PGFM responses.
Keywords: cattle; early pregnancy; oxytocin; PGF-2α
L. J. Jenner, T. J. Parkinson, and G. E. Lamming
Summary. Binding of [3H]oxytocin to uterine subcellular preparations ('oxytocin receptor concentrations') was measured in uterine tissue of heifers and multiparous dairy cows at various stages of the oestrous cycle and during early pregnancy. A method for the assay of ovine uterine oxytocin receptors was optimized for use on bovine tissue. Oxytocin receptor concentrations were increased in cyclic animals around the period of luteolysis and oestrus, rising on Day 15 in endometrium and on Day 17 in myometrium while pregnant animals showed no comparable rise. Receptor concentrations then declined on Day 3 after oestrus in myometrium and on Day 5 in endometrium. Some cyclic animals did not show the expected rise in receptors in the late luteal phase; these animals had abnormally high progesterone concentrations for this stage of the cycle. In animals slaughtered on Day 18 after oestrus and/or insemination which had low oxytocin receptor levels, plasma progesterone concentrations were consistently high; while all animals showing the late luteal phase elevation in receptor values had low progesterone concentrations. Oxytocin receptor and progesterone concentrations were negatively correlated (P < 0·05). These data support the hypothesis that oxytocin receptor level is a key factor in the process of luteolysis in cattle and that in pregnancy there is suppression of uterine oxytocin receptor at the expected time of luteolysis. We suggest that uterine oxytocin receptor levels are partly controlled by circulating steroid hormones and are suppressed during early pregnancy.
Keywords: uterus; oxytocin; receptors; cattle; oestrous cycle; pregnancy
H. D. Nicholson, T. J. Parkinson, and K. R. Lapwood
This study was performed to determine whether oxytocin or vasopressin affect the transport of spermatozoa from the epididymis of rams in vivo. Under general anaesthesia, cannulae were inserted into each ductus deferens and passed into the cauda epididymis of 24 Oxford Down cross rams and the luminal fluid was collected at 10 min intervals for 2–3 h. Animals were divided into seven groups and received either (i) 2 ml 0.9% saline, (ii) 10 μg oxytocin, (iii) 100 μg oxytocin, (iv) 100 μg oxytocin antagonist, (v) 300 μg oxytocin antagonist followed by 100 μg oxytocin, (vi) 100 μg vasopressin, or (vii) 100 μg vasopressin followed by 100 μg oxytocin, all by i.v. injection. The mass of fluid and number of spermatozoa in each 10 min sample was measured and the motility of the spermatozoa was assessed. Treatment with saline did not affect the mass or the number of spermatozoa in the fluid collected. Oxytocin at 10 μg significantly increased both the output of fluid and the number of spermatozoa by twofold. Oxytocin at 100 μg produced a greater increase in both fluid output and the number of spermatozoa within 10 min of administration of the peptide. Treatment with oxytocin antagonist had no immediate effect, but subsequently caused a significant reduction in both fluid output and the number of spermatozoa. Pretreatment with oxytocin antagonist inhibited the stimulatory effect of oxytocin. Vasopressin did not increase the number or concentration of spermatozoa in the fluid and appeared to decrease fluid output. No significant changes in the morphology or motility of the spermatozoa collected was observed in any of the samples. These data demonstrate that oxytocin has specific actions on the epididymis to increase sperm transport. They indicate that local oxytocin may be involved in regulating basal contractility of the cauda epididymidis and that augmentation by the peptide in the peripheral circulation, as occurs around the time of ejaculation, may promote a significant increase in the transport of spermatozoa into the vas deferens and ejaculate.
S. A. Grant, S. E. Long, and T. J. Parkinson
Two techniques for the preparation of boar spermatozoa for in vitro fertilization were studied: a simple washing procedure and centrifugation on a discontinuous Percoll gradient. Their respective effects on motility of spermatozoa were analysed by computer-assisted sperm analysis. The Percoll density gradient technique selected spermatozoa with significantly (P < 0.0001) enhanced motility and movement characteristics. In vitro matured oocytes inseminated with spermatozoa prepared by Percoll gradient centrifugation had significantly (P < 0.0001) greater cleavage rates than did oocytes inseminated with washed spermatozoa. This increased penetration ability was not due to an increased proportion of acrosome-reacted spermatozoa. Transmission electron microscopy revealed no unique ultrastructural differences between the spermatozoa from either preparation. Spermatozoa prepared by Percoll gradient centrifugation are recommended for insemination and studies of porcine in vitro fertilization.
K. R. Stevenson, T. J. Parkinson, and D. C. Wathes
Summary. Jugular venous blood samples were collected throughout a complete oestrous cycle from 9 mares for measurement of progesterone and oxytocin by radioimmunoassay. Mean oxytocin concentrations remained at ∼ 1 pg/ml throughout, with no evidence of cyclic variation in the release pattern. Extracts of corpus luteum and follicles obtained from a further 33 mares at different stages of the cycle all contained oxytocin concentrations of < 10 pg/g wet weight of tissue. We conclude that the ovaries are not a source of circulating oxytocin during the oestrous cycle in this species. The plasma oxytocin concentrations reported here are substantially lower than those found by other groups.
Keywords: oxytocin; mare; oestrous cycle
K Whittington, SJ Assinder, T Parkinson, KR Lapwood, and HD Nicholson
Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.
T. J. Parkinson, D. C. Wathes, L. J. Jenner, and G. E. Lamming
Summary. Concentrations of oxytocin were measured in corpora lutea obtained from heifers throughout the oestrous cycle and first 30 days of pregnancy. Values were low during the first 3 days of the cycle (<250 ng/g tissue), increasing to 1312 ng/g by Day 4. Values then further increased up to a maximum of 2344 ng/g on Day 12. Concentrations were similar in cyclic and pregnant animals throughout the midluteal phase and were maintained at ∼ 1500 ng/g until the 18th (cyclic cows) or 19th (pregnant cows) day after oestrus, when they were again low. Values subsequently remained <250 ng/g in pregnant cattle. Concentrations of oxytocin in jugular venous plasma of cyclic (n = 5) and pregnant (n = 4) cows were measured in samples collected every 15 min for 8 h on Days 14, 16, 18 and 19 after oestrus. There were no significant differences in mean concentrations (range: 2·5–4·7 pg/ml) or in the number, frequency or area under the curve of episodes between either cyclic and pregnant animals, or between days. Mean basal concentrations were higher on Day 16 than on Day 14 (P < 0·05), values on Days 18 and 19 being intermediate. These findings suggest that the corpus luteum contains a finite amount of releasable oxytocin, which is exhausted by Day 18–19 after oestrus, whether or not pregnancy occurs, and that there is no further accumulation of oxytocin in the animal during early pregnancy. The contribution of luteal oxytocin to jugular venous concentrations appears to be less than in sheep, in which values in the jugular vein closely parallel those within the corpus luteum.
Keywords: oxytocin; corpus luteum; early pregnancy; cattle