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Aleona Swegen, Benjamin J Curry, Zamira Gibb, Sarah R Lambourne, Nathan D Smith and R John Aitken

Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell–cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC–MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim of furthering our understanding of this cell's complex biological machinery. We were able to identify 9883 peptides comprising 1030 proteins, which were subsequently attributed to 975 gene products. Gene ontology analysis for molecular and cellular processes revealed new information about the metabolism, antioxidant defences and receptors of stallion spermatozoa. Mitochondrial proteins and those involved in catabolic processes constituted dominant categories. Several enzymes specific to β-oxidation of fatty acids were identified, and further experiments were carried out to ascertain their functional significance. Inhibition of carnitine palmitoyl transferase 1, a rate-limiting enzyme of β-oxidation, reduced motility parameters, indicating that β-oxidation contributes to maintenance of motility in stallion spermatozoa.

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R. J. Aitken, E. A. Rudak, D. W. Richardson, J. Dor, O. Djahanbahkch and A. A. Templeton

Summary. Anti-zona antibodies are effective inhibitors of fertilization in vitro and, regardless of whether passive or active immunization techniques are used, in vivo. Antibodies raised against unfractionated zona pellucida antigens are chiefly directed against a group of carbohydrate-rich components localized on the outer surface of the zona. The interaction of anti-zona antibodies with these sites induces the formation of a surface precipitate which occludes the sperm binding sites by a process of steric hindrance, and stabilizes the zona structure against digestion by the proteolytic enzymes of the sperm head. Active immunization studies indicate that the long-term induction of infertility without adverse side effects is feasible in both laboratory rodents and primates when the zona pellucida is used as a target.

Anti-sperm antibodies also exhibit a capacity for inhibiting fertilization in vivo and in vitro. To determine the most appropriate detection method to screen patients for anti-sperm antibodies several homologous and heterologous antisera were analysed by 5 different agglutination and immobilization techniques and then compared for their ability to inhibit the fertilizing capacity of human spermatozoa using the zona-free hamster egg penetration test. The results obtained with the Franklin-Dukes tube—slide test exhibited the closest correlation with the anti-fertility activity of a given antiserum; this activity could be amplified by the addition of complement to the medium. It is concluded that antibodies directed against the sperm head are responsible for limiting the fertilizing capacity of human spermatozoa in vitro and that it is these antibodies on which attention should be focused to unravel the role that immunological factors play in the aetiology of infertility in vivo.

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T Leahy, J P Rickard, R J Aitken and S P de Graaf

Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7±2.7% to 2.8±1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

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I. Wilmut, W. A. Ritchie, C. S. Haley, C. J. Ashworth and R. P. Aitken

Summary. A comparison was made of the rate and uniformity of development of embryos recovered from Meishan and European white sows. The time of ovulation was estimated to be 34·3 and 49·0 h after the onset of oestrus in large white and Meishan sows, respectively. Embryos were recovered from a total of 38 Meishan and 37 European pigs between 18 and 219 h after the estimated time of ovulation. Embryos recovered after 18–59 or 44–82 h were classified into one of 11 stages (from early fertilization to early blastocyst), and the maximum blastocyst diameter was measured for embryos recovered 140–219 h after ovulation. There was no evidence of a difference between the genotypes in the stage or size of embryos at these times or of large differences between the genotypes in the extent of variation in embryo stage within females, although a minority of European white females had very variable embryos. As the differences between the embryos of the Meishan and the European white were small, it seems unlikely that greater uniformity of Meishan embryo development is a major cause of the higher prenatal survival in that breed.

Keywords: pigs; prenatal survival; embryo development

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

The objective of this study was to investigate whether peripheral plasma profiles of pregnancy specific protein B (PSPB) are predictive of pregnancy outcome in adolescent sheep in which growth of the placenta has been compromised by the competing nutrient demands of maternal tissue synthesis. Embryos recovered on day 4 after oestrus from adult ewes inseminated by a single sire were transferred in singleton to prepubertal adolescent recipients. After transfer, the adolescent recipients were individually offered a high or low proportion of a complete diet to promote rapid (RMG) or normal (NMG) maternal growth rates (n = 12 per group). After day 100 of gestation the feed intake of the NMG group was adjusted weekly to meet the nutrient requirements of the gravid uterus. Blood was sampled three times a week throughout gestation and analysed for PSPB and progesterone. Liveweight gain during the first 120 days of gestation was 229 ± 9.1 and 105 ± 3.9 g day−1 for the RMG and NMG groups, respectively. For ewes delivering live young, mean placental mass at term was 263 ± 16.8 and 438 ± 44.6 g (P < 0.002), while lamb birthweight was 2.74 ± 0.25 and 4.34 ± 0.27 kg (P < 0.001) for the RMG (n = 8) and NMG (n = 11) groups, respectively. The biphasic pattern of PSPB secretion during gestation was similar in all ewes delivering live young, but individual concentrations within treatment groups were highly variable. Mean PSPB concentrations were lower in RMG than in NMG ewes throughout gestation (P < 0.05) and the major differences in relative terms were detected between days 50 and 100 of pregnancy. PSPB concentrations during this latter period were correlated (P < 0.05) with placental mass at term but not with lamb birthweight. High dietary intakes, leading to rapid maternal growth rates were associated with low peripheral progesterone concentrations (P < 0.02) throughout gestation. Irrespective of treatment group, progesterone concentrations during the second half of pregnancy were positively associated with both placental mass at term (P < 0.002) and lamb birthweight (P < 0.01). The incidence of non-infectious abortion during late gestation (125 ± 1.3 days) was higher (P < 0.001) in the RMG (4 of 12) than in the NMG (1 of 12) group and was associated with abnormal PSPB profiles in the former group. The mass of the fetus at the time of abortion was highly correlated (P < 0.01) with mean PSPB concentrations up to day 120 of gestation, but was independent of peripheral progesterone concentrations. These results suggest that sequential measurement of PSPB may provide a reliable indicator of fetal distress and adverse pregnancy outcome in singleton bearing ewes. PSPB and progesterone analysis may also have prognostic value as a biochemical marker of suboptimal placental growth and function in sheep.

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C. J. Ashworth, C. S. Haley, R. P. Aitken and I. Wilmut

Summary. Embryos were transferred between Meishan and Landrace × Large White (control) gilts on Day 4 or 5 to establish approximately equal numbers of all four possible combinations of donor breed and recipient breed. The breed of the donor gilt significantly (P < 0·01) affected embryo survival with 44·5% of transferred Meishan embryos and 69·6% of transferred control embryos surviving to Day 30 ± 1. There was no influence of the breed of the recipient gilt on the proportion of embryos which survived. These differences in embryo survival between the two breeds could not be explained by differences in (1) the number of embryos transferred, (2) the stage of development of the embryos transferred, (3) the interval between ovulation and transfer or (4) the degree of asynchrony between donor and recipient gilt.

On Day 30 ± 1 embryos from control donors developed into longer fetuses (P < 0·01) with larger allantoic sacs (P < 0·05) than did embryos from Meishan donors. Fetuses in control recipients were longer (P < 0·01), heavier (P < 0·001) and had larger allantoic sacs (P < 0·05) than fetuses occupying Meishan uteri. The interaction between breed of donor gilt and breed of recipient gilt did not significantly affect conceptus growth.

These results suggest that Meishan pig embryos may be less tolerant to routine embryo transfer procedures than those of control gilts, that the genotype of the dam does not affect the proportion of embryos surviving to Day 30 ± 1. and that both fetal and maternal factors affect conceptus growth.

Keywords: Meishan pig; embryo transfer; embryo survival; conceptus growth

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R. J. Aitken, D. Buckingham, K. West, F. C. Wu, K. Zikopoulos and D. W. Richardson

Summary. Cells isolated from the ejaculates of a high proportion of patients exhibiting oligozoospermia are characterized by generation rates of reactive oxygen species that considerably exceed those obtained for the normal fertile population. The purpose of this study was to resolve the cellular source of this enhanced activity. Semen samples from a cohort of oligozoospermic patients and a group of fertile controls were fractionated on discontinuous Percoll gradients to generate three cell populations (0, 50 and 100%) of differing density. For each fraction, both the steady-state and the phorbolester-induced chemiluminescent signals were significantly (P < 0·001) greater for the oligozoospermic samples than for the fertile controls. In the fertile donors, leucocytes comprised the major source of reactive oxygen species, particularly in the low-density Percoll fractions; in oligozoospermic patients, however, spermatozoa were identified as a second major source of reactive oxygen species. Particularly striking was an intense phorbol-ester-induced chemiluminescent signal generated by oligozoospermic spermatozoa, purified by passage through isotonic Percoll and free of leucocyte contamination, which was 167 times greater than the median signal generated by the corresponding fraction from the fertile controls (P < 0·001). These results emphasize the importance of spermatozoa as a major source of reactive oxygen species in oligozoospermia and have implications for the diagnosis and treatment of this condition, as well as for the design of appropriate diagnostic strategies.

Keywords: oligozoospermia; reactive oxygen species; spermatozoa; human

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T B Smith, G N De Iuliis, T Lord and R J Aitken

The discovery of a truncated base excision repair pathway in human spermatozoa mediated by OGG1 has raised questions regarding the effect of mutations in critical DNA repair genes on the integrity of the paternal genome. The senescence-accelerated mouse prone 8 (SAMP8) is a mouse model containing a suite of naturally occurring mutations resulting in an accelerated senescence phenotype largely mediated by oxidative stress, which is further enhanced by a mutation in the Ogg1 gene, greatly reducing the ability of the enzyme to excise 8-hydroxy,2′-deoxyguanosine (8OHdG) adducts. An analysis of the reproductive phenotype of the SAMP8 males revealed a high level of DNA damage in caudal epididymal spermatozoa as measured by the alkaline Comet assay. Furthermore, these lesions were confirmed to be oxidative in nature, as demonstrated by significant increases in 8OHdG adduct formation in the SAMP8 testicular tissue (P<0.05) as well as in mature spermatozoa (P<0.001) relative to a control strain (SAMR1). Despite this high level of oxidative DNA damage in spermatozoa, reactive oxygen species generation was not elevated and motility of spermatozoa was found to be similar to that for the control strain with the exception of progressive motility, which exhibited a slight but significant decline with advancing age (P<0.05). When challenged with Fenton reagents (H2O2 and Fe2 +), the SAMP8 spermatozoa demonstrated a highly increased susceptibility to formation of 8OHdG adducts compared with the controls (P<0.001). These data highlight the role of oxidative stress and OGG1-dependent base excision repair mechanisms in defining the genetic integrity of mammalian spermatozoa.

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R John Aitken, Jock K Findlay, Karla J Hutt and Jeff B Kerr

Apoptosis is a critical process for regulating both the size and the quality of the male and female germ lines. In this review, we examine the importance of this process during embryonic development in establishing the pool of spermatogonial stem cells and primordial follicles that will ultimately define male and female fertility. We also consider the importance of apoptosis in controlling the number and quality of germ cells that eventually determine reproductive success. The biochemical details of the apoptotic process as it affects germ cells in the mature gonad still await resolution, as do the stimuli that persuade these cells to commit to a pathway that leads to cell death. Our ability to understand and ultimately control the reproductive potential of male and female mammals depends upon a deeper understanding of these fundamental processes.

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L. M. Mitchell, M. E. King, R. P. Aitken, F. E. Gebbie and J. M. Wallace

The objective of this study was to determine the relative importance of seasonal changes in ovulation rate, fertilization rate and embryo survival as the cause of reduced lambing rates in ewes mated in February compared with those mated in November. The study was conducted at 57°N using mature Mule ewes and Suffolk rams. Sixty ewes were allocated equally to five groups: unbred (UB) or mated at a natural oestrus during November (N) or February (F) by natural (N) or cervical artificial (A) insemination. Groups were maintained separately at pasture supplemented with hay. A raddled vasectomized or non-vasectomized ram was present with UB, NN and NA groups from 26 October 1995 to 1 January 1996 and with UB, FN and FA groups from 25 January 1996 to 31 March 1996. Ewes marked by the ram were recorded twice a day, and those in groups NN, NA, FN and FA were inseminated at their second behavioural oestrus. For all ewes, blood samples were obtained once a day from introduction of the vasectomized rams until 30 days after mating (groups NN, NA, FN and FA) or 20 days after the first oestrus (group UB), and ovulation rate was measured by laparoscopy 7 days after the first oestrus. For ewes in groups NN, NA, FN and FA, ovulation rate was measured again after the second oestrus and ova were recovered from six ewes per group for assessment of fertilization before autotransfer. Pregnancy and lambing rates were recorded at term. Mean (± se) dates of the first recorded oestrus for ewes in groups NN, NA and UB, and FN, FA and UB were 4 ± 1.1 November and 4 ± 0.9 February, respectively, and intervals between the first and second oestrus were 16 ± 0.2 and 17 ± 0.3 days (P < 0.01), respectively. Ovulation rates were 2.6 ± 0.08 and 2.0 ± 0.05 (P < 0.001), and peripheral progesterone concentrations during the luteal phase were 8.5 ± 0.25 and 7.6 ± 0.31 ng ml−1 (P < 0.05), for November and February, respectively. The difference in peripheral progesterone concentration was not solely attributable to the difference in ovulation rate. There was no significant effect of month or method of insemination, or of embryo recovery and autotransfer procedures on pregnancy rates and the proportion of ewes that became pregnant were NN 0.92, NA 0.83, FN 0.67 and FA 0.75. For ewes undergoing embryo recovery and autotransfer, ova recovered per corpus luteum were 1.00, 0.93, 1.00 and 0.92, fertilized ova per ovum recovered were 0.69, 0.92, 1.00 and 0.83, and lambs born per corpus luteum were 0.62, 0.79, 0.78 and 0.58 for NN, NA, FN and FA groups, respectively. There were no significant seasonal effects on fertilization rate or embryo survival. It is concluded that a seasonal decline in ovulation rate is the primary cause of reduced lambing rates in ewes mated in February compared with those mated in November. Pregnancy rates were high after mating in both periods and were not enhanced by the use of cervical insemination.