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R. J. Aitken, D. Buckingham and D. Harkiss

The reaction between xanthine and xanthine oxidase results in the univalent and divalent reduction of dioxygen to generate superoxide (O2 −•) and hydrogen peroxide (H2O2), respectively. With the aid of this system, the direct effect of reactive oxygen species (ROS) on human sperm function has been investigated. A protocol involving the addition of xanthine oxidase to the reaction mixture at 0 and 15 min resulted in a loss of motility involving every component of sperm movement examined. Lower doses of xanthine oxidase, which did not influence sperm motility, were also found to suppress the competence of human spermatozoa to exhibit oocyte fusion in response to the ionophore, A23187. The reactive oxygen species responsible for the disruption of human sperm function was not influenced by the presence of superoxide dismutase (SOD) or scavengers of hypochlorous acid or hydroxyl radicals. However, the cytotoxic species was shown to be extremely stable and could be completely eliminated by catalase, which selectively eliminates H2O2. Confirmation that it is H2O2, and not O2 −•, which is cytotoxic to human spermatozoa was obtained in studies in which the direct addition of this oxidant was shown to influence both the movement of human spermatozoa and their competence for oocyte fusion. These results carry implications for the diagnosis of defective sperm function and the design of optimized culture media for the treatment of male factor infertility.

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R. J. Aitken and D. W. Richardson

Summary. Conditions have been defined under which the sperm binding capacity of the mouse zona pellucida can be measured. As a measure of the concentration of anti-zona antibodies the inhibition of sperm binding appears to be both repeatable and sensitive. Using this technique anti-zona antibody titres were monitored in 6 rats actively immunized with extracts of mouse ovarian tissue. These animals exhibited a rapid rise in anti-zona antibody titre following the induction of immunity, and an associated significant (P < 0·05) decline in their fertility, both in relation to the proportions of animals exhibiting fertile matings and of matings resulting in conception. Three of the animals exhibited permanent sterility despite repeated exposure to fertile males.

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C. J. Henderson, M. J. Hulme and R. J. Aitken

Summary. The notion of a contraceptive vaccine based on gamete-specific surface antigens was first proposed over a decade ago, as the result of in-vitro and in-vivo studies, and in recent years has been the subject of intensive research. In particular, the zona pellucida has attracted much attention as a potential target for immunological intervention in the fertilization process. Such is the rapidly-expanding nature of research into the biochemical and biological characterization of this structure, that a review of the implications for the development of a contraceptive vaccine seems timely.

Keywords: zona pellucida; contraceptive vaccine; antibodies

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J. M. Wallace, J. J. Robinson and R. P. Aitken

Summary. After lambing in late November, oestrus and ovulation were induced by using a CIDR device and PMSG in early weaned (N = 13) or lactating (N = 14) Border Leicester × Scottish Blackface ewes between 23 and 29 days after parturition. Ewes were intrauterine inseminated under laparoscopic visualization 54–55 h after CIDR-device withdrawal and eggs recovered on Day 3 of the cycle. Ovum recovery and fertilization rates were higher in lactating than in early weaned ewes, with fertilization being achieved as early as 24 days post partum in both groups. Of the 7 early weaned and 11 lactating ewes yielding eggs, fertilization occurred in 4 and 7 ewes respectively. A total of 20 embryos were transferred to the normal uterine environment of 15 recipient ewes in which the interval from parturition was > 150 days. Pregnancies were successfully established in 9 recipient ewes, resulting in the birth of 10 viable lambs.

Prolactin concentrations were significantly higher (P < 0·001) in lactating than in early weaned ewes throughout the study. Nevertheless, normal luteal function (as assessed by daily progesterone concentrations) was exhibited by 12 of 14 lactating and 8 of 13 early weaned ewes. Two post-partum donors in which the corpora lutea completely failed to secrete progesterone yielded fertilized eggs which developed to term when transferred to a normal uterine environment.

The results show that sheep oocytes can be fertilized using laparoscopic intrauterine insemination as early as 24 days after parturition and that the resulting embryos are viable when recovered on Day 3 after oestrus and transferred to a normal uterine environment.

Keywords: post partum; fertilization; embryo viability; pregnancy; sheep

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R. J. Aitken, S. Irvine and R. W. Kelly

Summary. The selective ability of PGE-1 and PGE-2 to enhance the capacity of human spermatozoa for hamster oocyte penetration was dependent upon high concentrations (1·7 mm) of extracellular Ca2+ and a prolonged (4 h) duration of exposure, and insensitive to the Ca2+ channel antagonist, verapamil. Studies with the intracellular calcium indicator Quin-2 indicated that exposure to PGE-1 and PGE-2, but not PGF-2α, induced a significant rise in the levels of cytoplasmic Ca2+, suggesting that an ionophore-like action might be responsible for the ability of the E-series prostaglandins to influence sperm function.

Stimulation of oocyte penetration with PGE-1 and PGE-2 was significantly enhanced when these compounds were presented to the spermatozoa in medium of high osmolarity (410 mosmol). A combination of PGE-1 in hyperosmotic medium did not significantly influence sperm function in cases of oligozoospermia, although it was effective with patients exhibiting idiopathic infertility.

Exposure to high doses of PGE-1 and PGE-2, but not 19-hydroxy PGE or PGF-2α, also induced a significant rise in the cyclic AMP content of human spermatozoa. This effect did not appear to be involved in the enhancement of fertilization rates because (1) it did not exhibit the same absolute dependence on high levels of extracellular Ca2+ as did the fertilization responses and (2) the enhancement of oocyte penetration and the elevation of cAMP were independent of each other within the patient population.

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Jacqueline M. Wallace, J. J. Robinson and R. P. Aitken

Summary. In Exp. 1 the effect of lactation versus early weaning on luteal function was examined in seasonally anoestrous Finn Dorset ewes that were induced to ovulate at 21 (N = 14) or 35 (N = 14) days post partum by using a CIDR device and PMSG. Prolactin concentrations were significantly higher (P < 0·001) in lactating compared with early weaned ewes throughout the study. The proportion of lactating ewes with inadequate luteal function (as assessed by daily progesterone concentrations) in the 21-day group was 0·43 (3 or 7) compared with 0·67 (4 of 6) for those weaned within 2 days after parturition. Corresponding values for the 35-day group were 0 (0 of 4) and 0·14 (1 of 7) respectively. There was no evidence of abnormal luteal function in standard ewes (N = 8) for which the interval from parturition was > 150 days.

In Exp. 2 we examined whether pregnancy can be successfully established during the breeding season following transfer of embryos into lactating or early weaned ewes in the early post-partum period. Embryos were donated from Border Leicester × Scottish Blackface ewes for which the interval from previous parturition was > 150 days. These embryos were transferred synchronously on Day 5 after behavioural oestrus to recipient ewes with the same breeding history as the donors (standard ewes, N = 15) or to lactating or early weaned recipients that had been induced to ovulate on Day 21 (N = 16) or 35 (N = 24) post partum. In the 21-day group inadequate luteal function was observed in 2 of 7 (0·28) lactating and 4 of 9 (0·44) early weaned ewes compared with corresponding values of 1 of 13 (0·08) and 2 of 11 (0·18) in the 35-day post-partum group. Luteal function was normal in all standard ewes. The proportion of successful pregnancies in the standard ewes was 0·80 (12 of 15) compared with 0 in lactating and early weaned ewes in the 21-day group and 0·08 (1 of 13) and 0·36 (4 of 11) respectively in the 35-day group.

The incidence of inadequate luteal function is therefore independent of the suckling stimulus and is higher in ewes induced to ovulate on Day 21 than Day 35 post partum during breeding and non-breeding seasons. For early post-partum recipient ewes with normal luteal function it is suggested that the high incidence of pregnancy failure after transfer of embryos may be due to embryo mortality caused by an inappropriate uterine environment or the inability of the embryo to sustain its luteotrophic signal.

Keywords: post partum; corpus luteum; embryo transfer; pregnancy; uterus; ewe

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J. J. Robinson, Jacqueline M. Wallace and R. P. Aitken

Summary. In Exp. 1, 40 ewes were used in a 2 × 2 factorial design to investigate the effects of intrauterine versus cervical insemination and superovulation using pig FSH or PMSG and GnRH on egg recovery and fertilization rate. Cervical inseminations were carried out at 48 and 60 h (N = 20 ewes) and intrauterine insemination at 52 h (N = 20 ewes) after progestagen pessary withdrawal. Eggs were recovered on Day 3 of the oestrous cycle. Ovulation, egg recovery and fertilization rates were independent of the type of superovulatory hormone used. Fertilization rate was high irrespective of insemination site but intrauterine insemination at 52 h was associated with a significant (P < 0·01) decrease in egg recovery of over 40% compared with cervically inseminated ewes.

In Exp. 2 ewes were inseminated at 36 (N = 5), 48 (N = 6) or 60 (N = 6) h after pessary withdrawal to determine the optimum intrauterine insemination time to maximize both fertilization rate and egg recovery. Egg recovery per ewe flushed was 23, 59 and 67% after intrauterine insemination at 36, 48 and 60 h respectively. Correspondingly, 0, 85 and 100% of the eggs recovered were fertilized. The results of Exps 1 and 2 suggest that when intrauterine insemination occurs before or during ovulation it interferes with oocyte collection by the fimbria.

In Exp. 3 egg recovery and fertilization rates were determined after cervical insemination at 48 and 60 h (N = 8) or intrauterine insemination at 48 (N = 9) or 60 (N = 8) h after progestagen withdrawal. Ewes in the last two groups were subdivided and inseminated unilaterally or bilaterally. Egg recovery was high after cervical insemination (95%) but only 36% of these eggs were fertilized. Unilateral intrauterine insemination was as effective as bilateral in ensuring high fertilization rates (100 versus 97%). Intrauterine insemination at 48 h compared with 60 h resulted in a significantly lower (P < 0·05) percentage of eggs recovered (42 versus 90% respectively). However, reducing the degree of interference by adopting unilateral rather than bilateral insemination did not alleviate the detrimental effects of the 48-h insemination time on egg recovery.

From these results we advocate the adoption of intrauterine insemination at 60 h after progestagen withdrawal to maximize fertilization rate and egg recovery in superovulated ewes.

Keywords: superovulation; intrauterine insemination; fertilization; ovum recovery; ewe

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The ultrastructure of four roe-deer blastocysts at different stages of embryonic development were studied. During delayed implantation, the outer surface of the trophoblast possessed numerous microvilli and periodic invaginations or caveolae. There was a marked lack of organelles such as mitochondria or endoplasmic reticulum in the cytoplasm of the trophoblast cells, though many lipid droplets, granular inclusions and a lamina of fine fibrillae were present. Elongation of the blastocyst was associated with a decrease in the size and number of the microvilli, the disappearance of lipid droplets and granular inclusions, a reduction in the amount of fibrillar material and a dramatic increase in the development of mitochondria, granular endoplasmic reticulum, ribosomes and Golgi apparatus.

The histology of the ovaries and uterus was studied in thirty-one roe deer. No prominent changes occurred in the ovaries at any stage of development; all ovaries possessed active CL and showed signs of follicular growth and atresia. Changes in the degree of mitotic activity, epithelial cell height, endometrial vascularity and stromal oedema were observed in the uterus throughout the period of delayed implantation and during the phase of rapid embryonic growth. Elongation of the embryo was associated with a marked decline in the height of the glandular epithelium and an increase in endometrial vascularity.

The most important ultrastructural changes in the uteri of six roe deer were observed in the endometrial glands. Delayed implantation was associated with the accumulation of numerous supranuclear vesicles derived from the Golgi apparatus, while the resumption of embryonic growth was correlated with their sudden disappearance. When elongation had been completed, there was a sudden decrease in the cellular activity of these endometrial glands.

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J. M. Wallace, R. P. Aitken and M. A. Cheyne

We examined the key events underlying maternal recognition of pregnancy and the prevention of luteolysis in early postpartum ewes by synchronously transferring single expanded blastocysts recovered from control ewes on day 11 of pregnancy into the uterus of either postpartum recipients that had been induced to ovulate 28 days after lambing (n = 12) or control recipients (n = 11). Conceptus development, uterine flush interferon (oTP-1) concentrations, endometrial oxytocin receptor concentrations and endometrial prostaglandin F (PGF) release in vitro were determined 5 days later (corresponding to day 16 of the ovarian cycle). By this stage, both conceptus mass and oTP-1 content of total uterine flush in the eight postpartum recipients that remained pregnant were significantly lower (P < 0.01) than in the eight pregnant control ewes (524 ± 116.6 versus 959 ± 80.6 mg and 968 ± 16.9 versus 1512 ± 106.2 ng oTP-1 for postpartum and control recipients, respectively). These effects were independent of ovulation rate and daily peripheral progesterone concentrations after blastocyst transfer, which were similar between groups. Endometrial oxytocin receptor density was variable in both groups when they were killed, and was generally higher in pregnant postpartum than in control recipients, and was significantly different (P < 0.05) when the values for the transfer but not the contralateral uterine horns were compared. Similarly, basal and oxytocin-stimulated endometrial PGF release during a 4 h incubation were higher (P < 0.01) in pregnant postpartum versus control recipients. Irrespective of treatment group and when expressed per uterine horn, conceptus mass was highly negatively correlated with number of oxytocin receptors and PGF release in vitro. The results of this study imply that suboptimal conceptus growth rates and secretion of oTP-1, resulting in an inability to regulate endometrial oxytocin receptor-mediated PGF secretion, may be central to pregnancy failure in the early postpartum ewe.

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R. J. Aitken, E. Holme, D. W. Richardson and M. Hulme

Summary. Intact and univalent antibodies were prepared against mechanically isolated mouse zonae pellucidae solubilized in a variety of ways (heat, low pH, SDS, urea and trypsin). The antisera bound avidly and specifically to solubilized iodinated zona antigens and the intact zona structure.

When the concentrations of immunoreactive Fab material in the intact and univalent antibody preparations were equalized and compared for their ability to block the sperm-binding stage of fertilization, only the intact γ-globulin preparations possessed antifertility activity. These results indicate that antibodies raised against intact solubilized zonae pellucidae block fertilization by cross-linking antigens on the outer zona surface, thereby indirectly masking the sperm receptor sites. The integrity of these surface components did not appear to be affected by solubilization procedures that disrupt non-covalent bonds (heating, low pH, SDS and urea) although they did appear to be adversely affected by trypsin treatment.

None of the antisera tested contained antibodies directed against the sperm receptor site indicating that these critical components lack immunogenicity.