A highly controlled model to investigate nutrient partitioning and the control of fetal growth in the rapidly growing adolescent sheep is described. Embryos recovered from superovulated adult ewes inseminated by a single sire were transferred in singleton to the uterus of prepubertal adolescent recipients induced to ovulate at 21 weeks of age (liveweight 44.4 ± 0.38 kg). After embryo transfer, the adolescent recipients were individually offered a high (n = 28) or low (n = 20) quantity of a complete diet calculated to achieve rapid (RMG) or normal (NMG) maternal growth rates. After day 100 of gestation the feed intake of the NMG group was adjusted weekly to meet the increasing nutrient demands of the gravid uterus. The proportion of adolescent recipients initially conceiving was significantly (P < 0.01) influenced by maternal nutrient intake and was lower in the RMG (0.57) than in the NMG (0.85) group. For adolescent dams that maintained their pregnancies, liveweight gain during the first 95 days of gestation was significantly (P < 0.001) higher in the RMG compared with the NMG group (234 ± 9.5 and 75 ± 5.0 g day−1, respectively). Rapid maternal growth rates were associated with a significant reduction in both fetal and placental weights as determined when the animals were killed on day 95 of gestation (n = 3 per group) or at term. For the RMG (n = 8) and NMG (n = 11) groups, respectively, mean lamb birthweights at term were 2.74 ± 0.25 and 4.34 ± 0.27 kg (P < 0.001), while term placental weights were 263 ± 16.8 and 438 ± 44.6 g (P < 0.002). The number of fetal cotyledons per placenta and mean fetal cotyledon weight were significantly lower in RMG compared with NMG ewes (P < 0.05). Irrespective of treatment group, lamb birthweight was highly positively correlated with placental weight and both parameters were negatively correlated with maternal liveweight gain during the first 100 days of gestation. The incidence of non-infectious spontaneous abortion at 125 ± 1.3 days of gestation was higher (P < 0.001) in the RMG (4 of 12) than in the NMG (1 of 12) group. Similarly, duration of gestation for those ewes delivering live young was shorter (P < 0.01) in the RMG compared with the NMG group (140 ± 0.94 versus 143 ± 0.28 days). Colostrum yield at parturition was positively related to placental weight and significantly lower (P < 0.001) in the RMG than in the NMG group (35 ± 12.1 and 247 ± 36.2 g, respectively). Neonatal survival rates at 72 h after parturition were reduced (P < 0.05) in the RMG (38%) compared with the NMG group (91%). These data suggest that in rapidly growing adolescent ewes, the established anabolic drive to maternal tissue synthesis is maintained at the expense of the gradually evolving nutrient requirements of the gravid uterus. This results in a major restriction in placental growth and a highly significant decrease in birthweight.
J. M. Wallace, R. P. Aitken and M. A. Cheyne
J Wallace, D Bourke, P Da Silva and R Aitken
Human adolescent mothers have an increased risk of delivering low birth weight and premature infants with high mortality rates within the first year of life. Studies using a highly controlled adolescent sheep paradigm demonstrate that, in young growing females, the hierarchy of nutrient partitioning during pregnancy is altered to promote growth of the maternal body at the expense of the gradually evolving nutrient requirements of the gravid uterus and mammary gland. Thus, overnourishing adolescent dams throughout pregnancy results in a major restriction in placental mass, and leads to a significant decrease in birth weight relative to adolescent dams receiving a moderate nutrient intake. High maternal intakes are also associated with increased rates of spontaneous abortion in late gestation and, for ewes delivering live young, with a reduction in the duration of gestation and in the quality and quantity of colostrum accumulated prenatally. As the adolescent dams are of equivalent age at the time of conception, these studies indicate that nutritional status during pregnancy rather than biological immaturity predisposes the rapidly growing adolescents to adverse pregnancy outcome. Nutrient partitioning between the maternal body and gravid uterus is putatively orchestrated by a number of endocrine hormones and, in this review, the roles of both maternal and placental hormones in the regulation of placental and fetal growth in this intriguing adolescent paradigm are discussed. Impaired placental growth, particularly of the fetal component of the placenta, is the primary constraint to fetal growth during late gestation in the overnourished dams and nutritional switch-over studies indicate that high nutrient intakes during the second two-thirds of pregnancy are most detrimental to pregnancy outcome. In addition, it may be possible to alter the nutrient transport function of the growth-restricted placenta in that the imposition of a catabolic phase during the final third of pregnancy in previously rapidly growing dams results in a modest increase in lamb birth weight.
R. J. Aitken, M. Sutton, P. Warner and D. W. Richardson
Summary. In a group of normospermic donors exhibiting hamster oocyte penetration scores of 0–100%, multiple regression analysis indicated that only 20% of the variation in fertilizing potential could be explained by differences in the movement characteristics of the spermatozoa following incubation in vitro. When the movement characteristics of the spermatozoa in semen were considered this figure was reduced to 6·8% as a result of significant differences in the motility patterns exhibited by the seminal and post-incubation sperm populations.
A much closer relationship was observed between the movement characteristics of human spermatozoa in semen and their ability to penetrate cervical mucus. When differences in motile sperm densities were taken into account, 76% of the variation in cervical mucus penetration could be accounted for by the existence of linear correlations with certain aspects of sperm movement (multiple R = 0·874). Of the various attributes of sperm motility measured (linear velocity of progression, frequency of rotation, amplitude of sperm head displacement, % rolling and % yawing), a failure to exhibit an adequate amplitude of lateral sperm head displacement was consistently found to be the most significant factor determining the success of sperm—cervical mucus interaction (R 2 = 0·53).
S. D. Kholkute, R. J. Aitken and S. F. Lunn
Summary. In adult animals the intramuscular injection of hCG was followed by a rapid rise in plasma testosterone levels within 2—3 h and at doses of 40 and 80 i.u. hCG this primary response was followed by a second peak of testosterone at 48 h. Prepubertal marmosets also responded to hCG stimulation with a rapid increase in plasma testosterone levels within 3 h, but the magnitude of this peak was lower than that observed in adult animals and no biphasic pattern was observed.
In adult and prepubertal animals a second dose of hCG (40 i.u.) administered 24 h after the initial injection failed to produce a further rapid increment in plasma testosterone levels.
D. W. Richardson, R. J. Aitken and N. B. Loudon
Summary. Progresssively motile spermatozoa were present in 42/47 (89%) of post-vasectomy specimens, and in 16 of 24 samples (66%) demonstrable fertilizing potential could be detected in the zona-free hamster egg assay. The mean ± s.e.m. post-vasectomy penetration rate of 20·6 ± 6·0% was, however, significantly lower (P < 0·001) than the pre-vasectomy value of 49·7 ± 6·4%. After vasectomy the concentration of motile spermatozoa in the ejaculates declined in a manner that was significantly correlated with the post-operative time interval. The spermatozoa also lost their potential for fertilization, although functionally competent spermatozoa were still being recovered from ejaculates produced 8 days after vasectomy.
These results emphasize the ability of human spermatozoa to retain some residual fertilizing potential when stored at core body temperature in sites distal to the epididymis, and suggest a role for functional tests in the management of the vasectomized male.
R. J. Aitken, M. J. Hulme, C. J. Henderson, T. B. Hargreave and A. Ross
Summary. Washed ejaculated human spermatozoa were surface labelled with 125I, using solid phase (iodogen) or enzymic (lactoperoxidase) methods, while membrane components possessing terminal galactose or galactosamine residues were labelled with the galactose oxidase–sodium [3H]borohydride technique. All three procedures revealed the presence of 2 major labelled surface components. The first comprised a broad band of radioactivity migrating just behind the ion front on SDS-PAGE, which could be extracted with chloroform and methanol, suggesting a lipid-like composition. The second fraction exhibited properties consistent with a major glycoprotein component of the human sperm plasma membrane, giving a peak of radioactivity with M r = 20 000, within which a discrete doublet of bands (M r = 17 000 and 19 000) could be resolved by autoradiography. A more detailed analysis of the labelled protein fraction after TCA precipitation revealed a number of other surface components, the major ones of which exhibited M r values of 30 000, 45 000, 66 000, 115 000 000 and 160 000.
Western blot analysis was then used to determine whether any of the surface components described above interacted with the γ-globulin fraction of antisera obtained from patients exhibiting idiopathic autoimmunity against sperm antigens. Using a purified membrane preparation as the target, antibodies were detected against numerous high molecular weight bands with M r values similar to the major components of the human sperm surface (35 000, 45 000, 66 000, 90 000 and 150 000). The nature of the antigens targeted by these antisera did not correlate with the ability of the latter to stimulate or suppress sperm–oocyte fusion.
J. J. Robinson, R. P. Aitken, T. Atkinson, J. M. Wallace and A. S. McNeilly
Twelve anoestrous ewes maintained under natural photoperiod at 57°N received an oral dose of 3 mg melatonin daily at 15:00 h from 1 May. Starting 41 days later and extending from 11 June until 5 September, six of the ewes were also infused continuously with 0.8 mg thyrotrophin-releasing hormone (TRH) day−1 via subcutaneous osmotic minipumps. The remaining six ewes acted as controls. Behavioural oestrus, ovulation rate and luteal function were determined by exposure to a vasectomized ram, laparoscopy and the measurement of progesterone in peripheral plasma, respectively. TRH infusion stimulated a sustained increase (P < 0.001) in plasma concentrations of thyroxine, tri-iodothyronine and prolactin (thyroxine: 158 ± 9.3 and 65 ± 7.7 nmol l−1 for TRH-infused and control ewes, respectively; tri-iodothyronine, 2.6 ± 0.12 and 1.1± 0.19 nmol−1 and prolactin, 57±12 and 11 ± 2 μg l−1). No ewes were in oestrus before the TRH infusion and the mean number of behavioural oestrous cycles per ewe during the infusion period was 1.3 ± 0.33 and 2.5 ± 0.34 for TRH-infused and control ewes, respectively (P < 0.05). Corresponding mean intervals from 1 May to the onset of the first luteal phase (progesterone > 1 ng ml−1) were 88 ± 8.9 and 79 ± 3.5 days (not significant). TRH infusion had no effect on the mean numbers of corpora lutea (1.7 ± 0.14 and 1.6 ± 0.20 for TRH-infused and control ewes, respectively), but was associated with a lower mean incidence of normal luteal phases (1.5 ± 0.43 versus 2.7 ± 0.21, P= 0.052). Abnormalities in luteal function included delayed initial expression, extended ovarian cycles, suprabasal periovulatory progesterone concentrations and protracted periods of low progesterone secretion between successive ovarian cycles. Thus continuous TRH infusion suppressed plasma prolactin, doubled the circulating concentrations of thyroxine and tri-iodothyronine, and was associated with a wide range of abnormalities in ovarian function and endocrine status, the nature of which varied between ewes.
J. J. Robinson, J. M. Wallace, R. P. Aitken and S. Wigzell
Summary. Forty-two Scottish Blackface ewes that lambed outdoors in March were removed from their lambs at the end of April and housed under natural daylength at 57°N. Treatments (n = 7 ewes per treatment) commenced on 1 May and comprised daily oral dosing at 15:00 h with 3 mg melatonin dissolved in water and ethanol (4:1, v/v) for 30, 60, 90, 120 or 150 days. Control ewes received the vehicle alone. Ovarian activity was assessed by laparoscopy at monthly intervals with an additional interim observation in mid-July. Blood was sampled three times a week by jugular venepuncture and assayed for progesterone, prolactin and follicle-stimulating hormone (FSH). Luteinizing hormone (LH) was determined in blood samples collected at 15 min intervals for 10 h on days 28, 60, 91, 119 and 150.
Thirty days of melatonin treatment delayed (P < 0·01) first ovulation by about 1 month (mean interval ± sem from 1 May to progesterone > 1 ng ml−1, 165 ± 4·5 days versus 132 ± 9·2 days for controls). None of the ewes that received melatonin for 60 days ovulated before the end of melatonin treatment, but subsequently six of them did; the mean interval from 1 May to increased progesterone concentration was 75 ± 1·2 days. All ewes receiving melatonin for 90, 120 and 150 days ovulated with corresponding mean intervals of 83 ± 2·7, 85 ± 1·3 and 87 ± 2·2 days, respectively (P < 0·001 compared with controls). Ewes treated for 60, 90 and 120 days returned to anoestrus 4–6 weeks after the end of melatonin treatment; for the 120 day treatment this coincided with the onset of oestrous cyclicity in control ewes.
Melatonin had no effect on the concentrations of FSH. Thirty days of melatonin treatment in May delayed the autumn decline in LH pulse amplitude and increase in pulse frequency, whereas treatment for 120 and 150 days advanced these changes to late July–early August. Prolactin concentrations declined to minimum values after 30 days of melatonin treatment, but began to increase again after about 100 days. After the treatments for 90, 120 and 150 days, prolactin increased to higher concentrations (P < 0·001) than in controls and failed to return to control values before sampling ended in late October, i.e. 95, 65 and 35 days later, respectively.
Keywords: melatonin; ovulation; luteinizing hormone; prolactin; ewe
J. J. Robinson, J. M. Wallace, R. P. Aitken and A. S. McNeilly
Thirty-two Scottish Blackface ewes that lambed outdoors in March and were weaned at the end of April and individually penned indoors under the natural photoperiod at 57°N were used to determine whether the ovine ovary that was deprived of gonadotrophic support was capable of early activation by melatonin. From 5 May (day 0), 16 of the ewes received an oral dose of 3 mg melatonin in a 4:1 (v:v) mixture of water and ethanol daily at 15:00 h. The remaining 16 ewes received the vehicle alone. Within each of these groups, eight were implanted s.c. on day 0 with an osmotic minipump which infused 50 μg of the gonadotrophin releasing hormone agonist (GnRHa), buserelin day−1. On day 25, a second minipump was inserted to ensure continued infusion of the agonist and on day 50 (24 June) both minipumps were removed. Ovarian activity was assessed by laparoscopy at intervals of 3 weeks from day 29 until the experiment was terminated on day 200 (21 November). Blood samples taken by jugular venepuncture three times a week for the first 50 days, daily from days 51–78 and thereafter three times a week were analysed for progesterone, prolactin and LH. Samples taken at intervals of 15 min for 10 h on days −1, 14, 28, 49, 56, 70 and 91 were assayed for LH. Treatment with GnRHa reduced LH concentrations and abolished pulsatile LH secretion. The onset of ovarian activity (progesterone >3.8 nmol l−1) was not affected by the 50-day GnRHa treatment and occurred for the melatonin-treated ewes at mean (± sem) intervals from 5 May of 66 ± 2.9 (range 51–75) and 71 ± 0.9 (range 68–75) days for non-GnRHa and GnRHa ewes, respectively. For the ewes not receiving melatonin the corresponding intervals were 113 ± 11.6 and 125 ± 9.2 days, respectively. The mean numbers of corpora lutea at first oestrus were not affected by GnRHa treatment and were 1.4 ± 0.13 and 1.5 ± 0.13 for control and melatonin-treated ewes, respectively. First ovulation following GnRHa treatment resulted in luteal concentrations of progesterone of normal duration and magnitude but, within the melatonin-treated ewes, those that received GnRHa returned to anoestrus 21 days earlier (P < 0.03) than did their non-GnRHa counterparts. After their initial suppression by melatonin, prolactin concentrations began to increase after 80 days of melatonin treatment in both non-GnRHa and GnRHa ewes and approximately three months before the ewes returned to anoestrus. The results demonstrate that a period of isolation of the ovaries of the ewe from the pituitary gonadotrophins does not alter their ability to respond to the melatonin-induced activation of the GnRH pulse generator.
R. J. Aitken, F. Best, D. W. Richardson, R. Schats and G. Simm
Summary. When added to frozen–thawed human semen, the 3 doses of caffeine tested (2, 5 and 10 mM) induced a significant increase in the percentage of motile spermatozoa but did not influence the quality of movement. Considerable variability was noted between samples in their responsiveness to caffeine which, at the 5 and 10 mM doses, was significantly correlated with the degree of motility lost during cryostorage. Caffeine treatment of frozen–thawed human spermatozoa also increased the number of spermatozoa penetrating cervical mucus in unit time, by increasing the frequency rather than the success of collisions between spermatozoa and the cervical mucus interface. When caffeine-stimulated spermatozoa were washed free of seminal plasma containing this compound they were no longer at an advantage with respect to their motility or fertilizing ability. When 2 mM-caffeine was added to washed suspensions of capacitated spermatozoa it failed to stimulate motility but did significantly enhance the fertilizing ability of the spermatozoa, indicating a possible clinical role for this compound in in-vitro fertilization therapy.