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J. G. AALBERS, T. MANN and C. POLGE

Summary.

The metabolism of boar semen differs in several respects from that of bull and ram. Anaerobically, boar spermatozoa convert fructose to lactic acid at a much lower rate than ram or bull spermatozoa, and under these conditions their motility is also characteristically small. Aerobically, boar spermatozoa convert fructose to lactic acid at the same or even lower rate, yet their motility is very high. This is due to the ability of boar spermatozoa to oxidize lactic acid with an efficiency as high as that encountered in ram or bull semen. Apart from lactic acid, boar spermatozoa are capable of utilizing aerobically a number of other substrates, including glycerol, pyruvic acid and acetic acid, but unlike ram spermatozoa, they lack the ability to oxidize sorbitol. Two other unusual features of the aerobic metabolism of boar semen are (i) an increase in oxygen uptake, which occurs after the semen has been stored in vitro, and (ii) the occurrence in the boar seminal plasma of an easily oxidizable substance which is responsible for the plasma's own oxygen uptake.

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G. E. Mann, G. E. Lamming and J. H. Payne

The effects of the pattern and concentration of early luteal phase progesterone on subsequent prostaglandin F release in response to exogenous oxytocin was investigated during simulated luteal phases in ovariectomized cows treated with progesterone and oestradiol in patterns designed to simulate the range of luteal phase concentrations that occur naturally. In the first experiment, three groups of four cows received different concentrations of early luteal phase progesterone to determine the effective concentration in terms of cycle control. The results show that a plasma progesterone concentration early in the luteal phase as low as 0.6 ng ml−1 was sufficient to affect the timing of the subsequent luteolytic signal. In the second experiment, an early (day 1), a normal (day 4) or a late (day 7) postovulatory increase in progesterone was recreated in three groups of four cows. Responsiveness to oxytocin in the early progesterone group developed 3 days earlier than in the normal progesterone group, demonstrating the ability of early progesterone to advance the luteolytic signal. However, in the late progesterone group, there was no delay in the development of responsiveness to oxytocin compared with the normal progesterone group, demonstrating that the luteolytic signal is programmed to occur by a given time, irrespective of the early progesterone pattern. This demonstrates that a factor other than the timing of the early luteal phase progesterone increase ultimately must control the timing of luteolysis in cows.

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J. H. Hyland, J. G. Manns and W. D. Humphrey

Summary. For Day-14 sheep embryos, mean (±s.e.m., n = 7) PGE release was 20·2 ± 5·0, 13·0 ± 1·9 and 6·7 ± 1·9 ng.mg tissue−1.h−1 after 0·5, 4·5 and 8·5 h of culture respectively, while mean PGF release rates (±s.e.m.) for the same periods were 98·5 ± 24·4, 49·3 ± 16·3 and 16·5 ± 5·4 ng.mg tissue−1.h−1, respectively. Total quantities of PGE and PGF released after 8·5 h of culture were 707 ng and 1833 ng per embryo, respectively. Embryos (n = 21) collected between Days 13 and 15 of gestation contained 25·4±6·9 ng PGE and 66·2 ± 23·2 ng PGF/ embryo. A total PG release 28 times the embryonic content of PG suggested active PG production by the embryos. Co-culture of embryos with endometrium resulted in PG release rates which were not significantly higher than those from embryos cultured alone. Release of PGE and PGF from embryos declined with time, that for PGF being significantly (P < 0·05) reduced after 8·5 h incubation. Co-culture of embryos and endometrium had no effect on rate of decline of PG production over time. Day 14 endometrium from pregnant or non-pregnant ewes produced very small quantities of both PGs by comparison with embryos, and there was no consistent trend for PG production rate over time.

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R. Medhamurthy, T. D. Carruthers and J. G. Manns

Summary. Experiments were conducted with ewes to investigate the effects of an enriched bovine follicular fluid inhibin preparation (INH) on gonadotrophin secretion after the onset of oestrus. Administration of INH (10 mg) 1 h after the onset of oestrus did not significantly alter the preovulatory FSH and LH surges or the second FSH peak. To determine the effects of INH on the second FSH surge, ewes were treated with saline (N = 7) or INH (N = 10) at 4 h (10 mg) and 24 h (5 mg) after the peak of the preovulatory LH surge. The second FSH surge was delayed about 24 h (P < 0·05) in ewes treated with INH; however, the delay did not alter the interval to the next oestrus.

In a third experiment, 16 ewes were assigned to 4 groups in a 2 × 2 factorial with the main effects being ovariectomy at 4 h and INH treatment (10 mg) at 4, 20 and 36 h after the peak of the LH surge. Controls received sham ovariectomy and saline injection as appropriate. Ovariectomy resulted in a rapid increase in serum FSH but not LH and this was delayed (P < 0·05) by INH treatment. These results indicate that inhibin has a selective inhibitory action on FSH secretion in ewes and suggests that the second FSH surge results from increased basal FSH secretion due to decreased endogenous inhibin levels.

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R S Robinson, A J Hammond, G E Mann and M G Hunter

Luteal inadequacy is a major cause of poor embryo development and infertility. Angiogenesis, the formation of new blood vessels, is an essential process underpinning corpus luteum (CL) development and progesterone production. Thus, understanding the factors that regulate angiogenesis during this critical time is essential for the development of novel strategies to alleviate luteal inadequacy and infertility. This study demonstrates the development of a physiologically relevant primary culture system that mimics luteal angiogenesis. This system incorporates all luteal cell types (e.g. endothelial, steroidogenic cells, fibroblasts and pericytes). Using this approach, endothelial cells, identified by the specific marker von Willebrand factor (VWF), start to form clusters on day 2, which then proliferate and develop thread-like structures. After 9 days in culture, these tubule-like structures lengthen, thicken and form highly organized intricate networks resembling a capillary bed. Development of the vasculature was promoted by coating wells with fibronectin, as determined by image analysis (P<0.001). Progesterone production increased with time and was stimulated by LH re-enforcing the physiological relevance of the model in mimicking in vivo luteal function. LH also increased the area stained positively for VWF by twofold (P<0.05). Development of this endothelial cell network was stimulated by fibroblast growth factor 2 and vascular endothelial growth factor A, which increased total area of VWF positive staining on day 9, both independently (three- to fourfold; P<0.01) and in combination (tenfold; P<0.001). In conclusion, the successful development of endothelial cell networks in vitro provides a new opportunity to elucidate the physiological control of the angiogenic process in the developing CL.

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R. Webb, G. E. Lamming, N. B. Haynes, H. D. Hafs and J. G. Manns

Summary. Doses of 125, 250 or 500 μg LH-RH were injected i.m. into suckled beef cows on approximately Day 11 of an oestrous cycle synchronized by prostaglandin treatment. There was a positive linear relationship between dose of LH-RH and the area under the measured LH peak. Administration of 500 μg LH-RH as a single injection to suckled cows 13–32 days post partum resulted in LH release but failed to induce normal ovarian activity. A small transient rise in plasma progesterone for 6–9 days occurred at the expected time after injection in 50% of animals. Administration of 500 μg LH-RH to suckled beef cows approximately 20–30 days post partum and a second injection approximately 10 days later at the time when the resulting transient rise in plasma progesterone had returned to basal values induced normal cyclic activity (as shown by progesterone concentrations and observed oestrus) at 35 days compared with 70 days for untreated controls. Pituitary responsiveness to LH-RH, as assessed by LH levels, was found to increase up to 20 days post partum.

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R S Robinson, K J Woad, A J Hammond, M Laird, M G Hunter and G E Mann

Ovarian function is dependent on the establishment and continual remodelling of a complex vascular system. This enables the follicle and/or corpus luteum (CL) to receive the required supply of nutrients, oxygen and hormonal support as well as facilitating the release of steroids. Moreover, the inhibition of angiogenesis results in the attenuation of follicular growth, disruption of ovulation and drastic effects on the development and function of the CL. It appears that the production and action of vascular endothelial growth factor A (VEGFA) is necessary at all these stages of development. However, the expression of fibroblast growth factor 2 (FGF2) in the cow is more dynamic than that of VEGFA with a dramatic upregulation during the follicular–luteal transition. This upregulation is then likely to initiate intense angiogenesis in the presence of high VEGFA levels. Recently, we have developed a novel ovarian physiological angiogenesis culture system in which highly organised and intricate endothelial cell networks are formed. This system will enable us to elucidate the complex inter-play between FGF2 and VEGFA as well as other angiogenic factors in the regulation of luteal angiogenesis. Furthermore, recent evidence indicates that pericytes might play an active role in driving angiogenesis and highlights the importance of pericyte–endothelial interactions in this process. Finally, the targeted promotion of angiogenesis may lead to the development of novel strategies to alleviate luteal inadequacy and infertility.

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N. B. Haynes, H. D. Hafs, K. Purvis and J. G. Manns

Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough LE12 5RD, Leicestershire, U.K.

As part of a study on pituitary-testicular feedback mechanisms, the plasma hormone levels of 9 Friesian bulls, 6-8 months old, were examined at the time of unilateral castration. Blood (10 ml) was taken from indwelling jugular cannulae at 30 min intervals for 48 hr, a testis being removed under local anaesthesia (Lignocaine, Pharmaceutical Man. Co.) from each animal after 24 hr. Testosterone concentrations in plasma were measured by the radioimmunoassay method of Haynes, Hafs, Waters, Manns & Riley (1975) and LH concentrations by the radioimmunological procedure described by Oxender, Hafs & Edgerton (1972). One bull had no detectable testosterone after unilateral castration and was not included in the analysis.

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A summary of the results for 8 animals is given in Table 1. Hormone concentrations before castration were in accord with previous

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G. B. Mann, K. J. Fowler, D. Grail and A. R. Dunn

In this study a rapid, simple and inexpensive procedure is described which allows potential germ-line male mice to be identified with confidence. Spermatozoa recovered by uterine washing following mating with normal female mice was analysed in two ways. First, the patterns of expression of the different isoforms of glucose phosphate isomerase were determined. Since the glucose phosphate isomerase isoforms expressed in embryo stem (ES) cell lines are frequently different from those associated with the host blastocyst, it is possible to determine the proportion of spermatozoa produced by an individual animal that was of ES cell or host–blastocyst origin. Second, DNA of spermatozoa was subjected to polymerase chain reaction (PCR) analysis using primers with specificity for the targeted mutation in the ES cells. The PCR analysis was particularly valuable in identifying germ cell chimaeras in which the contribution of ES-derived spermatozoa was significantly less than that specified by the host blastocyst.

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Kathryn J Woad, Morag G Hunter, George E Mann, Mhairi Laird, Amanda J Hammond and Robert S Robinson

Fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF) A are thought to be key controllers of luteal angiogenesis; however, their precise roles in the regulation and coordination of this complex process remain unknown. Thus, the temporal and spatial patterns of endothelial network formation were determined by culturing mixed cell types from early bovine corpora lutea on fibronectin in the presence of FGF2 and VEGFA (6 h to 9 days). Endothelial cells, as determined by von Willebrand factor immunohistochemistry, initially grew in cell islands (days 0–3), before undergoing a period of vascular sprouting to display a more tubule-like appearance (days 3–6), and after 9 days in culture had formed extensive intricate networks. Mixed populations of luteal cells were treated with SU1498 (VEGF receptor 2 inhibitor) or SU5402 (FGF receptor 1 inhibitor) or control on days 0–3, 3–6 or 6–9 to determine the role of FGF2 and VEGFA during these specific windows. The total area of endothelial cells was unaffected by SU1498 treatment during any window. In contrast, SU5402 treatment caused maximal reduction in the total area of endothelial cell networks on days 3–6 vs controls (mean reduction 81%; P<0.001) during the period of tubule initiation. Moreover, SU5402 treatment on days 3–6 dramatically reduced the total number of branch points (P<0.001) and degree of branching per endothelial cell island (P<0.05) in the absence of changes in mean island area. This suggests that FGF2 is a key determinant of vascular sprouting and hence critical to luteal development.