An 125I-labelled calmodulin gel overlay procedure was used to direct calmodulin-binding proteins in bovine spermatozoa and seminal plasma. Several calmodulin-binding proteins with molecular masses ranging from 12 to > 200 kDa were detected in epididymal and ejaculated spermatozoa. Certain of these proteins exhibited preferential calmodulin-binding in the presence of Ca2+, while others exhibited binding only in its absence. In seminal plasma, only two major proteins with molecular masses of 15 and 16 kDa showed a higher calmodulin-binding activity in the presence of Ca2+, whereas several polypeptides in the range of 6–17 kDa bound higher amounts of radiolabelled calmodulin in the absence of Ca2+. Our previous study has shown that a group of closely related major proteins, designated as BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa, isolated from bovine seminal plasma (BSP) have molecular masses in the range of 15–30 kDa. This prompted us to investigate whether these polypeptides from bovine seminal fluid interact with calmodulin. The results indicated that calmodulin binds to purified BSP-A1, -A2, -A3 and BSP-30 kDa proteins in the presence and absence of Ca2+. Furthermore, many polypeptides of low molecular mass (6–14 kDa) in bovine seminal plama that crossreact with these BSP proteins also show high calmodulin-binding activity, particularly in the absence of calcium. This was further demonstrated following the limited proteolysis of the BSP proteins. Several tryptic-peptides of BSP-A1/-A2 and BSP-30 kDa exhibited higher calmodulin-binding activity than the intact BSP proteins. In view of the key role of Ca2+ in triggering the acrosome reaction and the role of calmodulin in intracellular transport of calcium, it is suggested that BSP proteins are involved in sperm capacitation and the acrosome reaction.
P. Manjunath, L. Chandonnet, L. Baillargeon and K. D. Roberts
B. A. Keel, B. W. Webster and D. K. Roberts
Summary. Ejaculates (164) were obtained from 17 donors serving on an artificial insemination by donor panel. Semen analysis was performed before and after freezing by an integrated microcomputerized system employing the multiple-exposure photography (MEP) method. Sperm count, motility, velocity, motility index (MI; product of the sperm velocity and percentage of motile spermatozoa) and motile density (MD) were determined for each ejaculate. After the initial evaluation the ejaculates were frozen in liquid nitrogen, thawed 24 h later, and assessed for post-thaw motility, velocity, MI and MD. The mean ± s.e. sperm count and volume for this group of donors was 148 ± 4 × 106/ml and 3·1 ± 0·1 ml, respectively. Mean ± s.e. values obtained from the prefreeze analysis were: motility = 64 ± 1%, velocity = 30 ± 0·4μm/sec, MI = 19 ± 0·5 μm/sec and MD = 94 ± 3 × 106/ml. Post-thaw analysis revealed a significant reduction (P < 0·01 in all values measured. Motility was reduced to 27 ± 1%, MI was reduced to 5 ± 0·3 μm/sec, and MD was reduced to 33 ± 1 × 106/ml Velocity was the least affected by cryopreservation, being reduced to 21 ± 0·5 μm/sec (P < 0·01). Cryopreservation resulted in a marked shift in the frequency distribution of sperm motility and motility index towards subnormal values while in the majority of ejaculates velocity and motile density were maintained in the normal range. Significant differences were noted amongst donors in the percentage change of the various semen measures as a result of cryopreservation. When within-subject coefficients of variation were calculated, velocity was the least variable parameter. These results indicate that, while cryopreservation results in significant reductions in the number of motile spermatozoa in the ejaculate, the velocity is only marginally reduced.
T. K. ROBERTS, J. C. BOURSNELL and A. D. BROWN
Opalescence and precipitation of boar seminal plasma proteins, specifically promoted by zinc, have been shown to be due to a zinc-precipitable protein (ZPP). The properties of this purified protein cannot be distinguished from those of the seminal haemagglutinin and their identity is postulated. The response of seminal plasma opalescence to low temperature is especially marked between 14°C and 6°C. This is notably similar to the already recorded response of sperm survival to cooling. When semen is cooled to 4°C, ZPP, accompanied by zinc, is absorbed by spermatozoa.
K Hardy, C Wright, S Rice, M Tachataki, R Roberts, D Morgan, S Spanos and D Taylor
The advent of human in vitro fertilization (IVF) over 30 years ago has made the oocyte and preimplantation embryo uniquely accessible. This accessibility has given rise to new micromanipulation techniques, such as intracytoplasmic sperm injection for treatment of male infertility, as well as embryo biopsy for preimplantation diagnosis of both genetic disease and aneuploidy, a major cause of early embryo demise and miscarriage. In the UK, average pregnancy rates after IVF and embryo transfer are < 25%, even after transfer of several embryos. Unfortunately, a third of these pregnancies involve multiple gestations. Research is currently focusing on methods to improve IVF success rates while reducing twin and triplet pregnancies and their associated increased morbidity and mortality. One approach is to develop screening methods to identify the most viable embryos, so that transfer of fewer healthy embryos will result in a higher proportion of singleton pregnancies. Screening methods include optimizing culture conditions for prolonged culture and selection of viable blastocysts for transfer, or embryo biopsy and aneuploidy screening. Assisted reproduction is also increasingly important in other branches of medicine: survival rates for cancer sufferers are improving continually and there is now a significant need for approaches to preserve fertility after sterilizing chemo-and radiotherapy treatment. Techniques for cryopreserving male and female gametes or gonadal tissue are being developed, although systems to grow and mature these gametes are in their infancy. Finally, there are also concerns regarding the safety of these new assisted reproductive technologies.
N. Lepage, P. Miron, R. Hemmings, K. D. Roberts and J. Langlais
The distribution of lysophosphatidylcholine, lyso-platelet-activating factor and platelet-activating factor (PAF) was studied in human plasma and in follicular and peritoneal fluid. In plasma, peritoneal and follicular fluids, 51%, 87% and 89%, respectively, of the total lipids were found in the protein fraction (the density > 1.21 fraction). Two forms of lyso-phospholipids were identified in this fraction: one of high affinity and one of low affinity for albumin. The metabolism of PAF in human follicular fluid, peritoneal fluid and plasma was also investigated. PAF-acetylhydrolase activity was found in both peritoneal and follicular fluids which induced a time-dependent hydrolysis of [3H]PAF. The half-life of PAF was estimated to be 7–12 min in plasma, 15–25 min in peritoneal fluid and approximately 2 h in follicular fluid. PAF-acetylhydrolase activity in embryo culture media supplemented with 10% serum was markedly inhibited by addition of commercial serum albumin. When 25 g albumin l−1 was added, 22% of [3H]PAF was hydrolysed h−1 compared with 72% in media without albumin. The concentrations of lysophosphatidylcholine measured in plasma, in follicular and peritoneal fluids were 252, 286 and 53 μmol l−1, respectively. The distribution of these lysophospholipids and the metabolism of PAF in the female genital tract fluids reported in the present study provide evidence for the involvement of these biologically active lipid mediators in a variety of reproductive processes including sperm–egg interactions and embryonic development.
P. Ross, N. Vigneault, S. Provencher, M. Potier and K. D. Roberts
Galactosyltransferase activity has been partially characterized in human seminal plasma. K m values of 130 μmol l−1 for UDP-galactose and 2.25 mmol l−1 for N-acetylglucosamine were calculated and the enzyme was found to be dependent on temperature and manganese and present as a highly active component of human seminal plasma. Galactosyltransferase was inhibited by nucleotides, glycosylated nucleotides, bovine and human α-lactalbumin but not by monosaccharides. Radiation inactivation studies revealed that the biologically active unit of seminal plasma galactosyltransferase has a molecular mass of 45 kDa. Although the majority of galactosyltransferase activity found in seminal plasma is probably of prostatic origin, we report for the first time that it is also present in human epididymal intraluminal fluid. Low activity was detected in the proximal caput region but activity increased to maximum values in the adjacent downstream segment, the intermediate caput region. Specific activity was relatively constant albeit at a lower value in the following epididymal segments and vas deferens. The significance of the epididymal and seminal plasma galactosyltransferase activities is unknown, but the enzyme could be implicated in glycosylation events that are known to be important in gamete interaction.
K. J. McDowell, D. C. Sharp, A. T. Fazleabas and R. M. Roberts
Summary. Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with l-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography.
Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (M r) 30 000–40 000 (pI values 4·5–5·5 and 6–7), one group of M r ∼22 000 (pI 6·5–7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with M r 20 000 (pI 5–6).
The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12–16 of dioestrus or Days 12–18 of pregnancy. Mares from Day 39 through 100 of pregnancy made up the third group.
Keywords: embryo; horse; proteins; endometrium; electrophoresis
M. C. Léveillé, K. D. Roberts, S. Chevalier, A. Chapdelaine and G. Bleau
Summary. Using an immunofluorescence technique on ovarian sections, zona-immunoreactive components were detected in the cytoplasm of the oocyte from the beginning of its growth, when it is surrounded by only a thin squamous follicular cell layer, up to the end of its growth. In parallel with oocyte growth, the staining intensity decreased in the ooplasm. No staining was observed in the cytoplasm of the granulosa cells during normal follicular development in adult cyclic females. However, staining of the granulosa cells was observed at some stages of follicular development in immature females. This staining was especially evident in the ovaries of immature females (22 or 26 days old) stimulated with PMSG. In addition, the staining of the granulosa cells was consistently observed in ovaries showing an abnormal histology. Increased staining of the zona at its outer and inner regions could be distinguished in normal follicles, but when staining occurred on the granulosa cells no such pattern was observed over the zona matrix. These studies indicate that the oocyte itself but not the granulosa cells elaborates the native immunogenic material of the zona pellucida. The administration of PMSG at particular stages of ovarian differentiation interferes with follicular development leading to an abnormal extracellular assembly of the zona and its degradation (phagocytosis) by the surrounding granulosa cells.
T. K. Schalue-Francis, P. W. Farin, J. C. Cross, D. Keisler and R. M. Roberts
Summary. In Exp. 1 twice daily i.m. injections of 2 mg recombinant bovine IFN-αI1 (rboIFN-αI1) (N = 24) or placebo (N = 25) were administered to ewes from Day 12 to Day 16 during a normal oestrous cycle. Treatment did not increase (P > 0·10) oestrous cycle length (20·7 ± 1·2 versus 18·5 ± 1·4 days). In Exp. 2, ewes were injected twice daily with 2 mg IFN (N = 34) or placebo (N = 36) from Days 11 to 18 after natural mating. The rboIFN-αI1 significantly (P = 0·05) improved pregnancy rate (79% versus 58%) as determined by a failure of ewes to return to oestrus within 50 days. The number of ewes that lambed was greatest in the rboIFN-αI1-treatment group (71% versus 50%; P = 0·07), and no teratogenic effects were observed in the young born to IFN-treated ewes. The study was repeated a second year with a more fecund group of ewes (Exp. 3). More (P = 0·08) ewes injected with rboIFN-αI1 (58/65) than placebotreated ewes (48/61) were judged pregnant by ultrasound. Again more ewes lambed (55 versus 45) and more lambs were born (98 versus 80) from the rboIFN-αI1-treated group. Combining the data from both studies revealed a significant (P = 0·01) effect of treatment. The amount of antiviral activity in jugular vein blood of ewes injected with rboIFN-αI1 (2 mg) was determined over time in Exp. 4. Activity rose to a maximum (∼450 IRU/ml) within 1–2 h and declined by over 75% in 24 h. Single injections of 1, 2 and 5 mg in buffer or 2 mg emulsified in sesame oil all gave similar profiles of antiviral activity in jugular blood over a 48-h period. In Exp. 5, antiviral activity was measured in uterine vein, ovarian artery and jugular vein serum of untreated pregnant (N = 7) and non-pregnant (N = 11) ewes at Day 15 after mating. Activity was detected in the uterine vein (58 ± 19 IRU/ml) of all pregnant ewes. The observations in Exps 1–5 are consistent with a role for conceptus-derived IFN-α in maternal recognition of pregnancy and suggest that supplemental IFN-α might be useful in improving pregnancy success in sheep.
Keywords: α-interferon; oestrous cycle; pharmacokinetics; pregnancy; sheep
G. W. Smith, P. C. Gentry, B. Bao, D. K. Long, R. M. Roberts and M. F. Smith
Extensive extracellular matrix remodelling occurs within the lifespan of the corpus luteum, particularly during corpus luteum formation and regression. A major mechanism for the regulation of extracellular matrix remodelling is via local production of specific proteinase inhibitors, such as the serine proteinase inhibitor plasminogen activator inhibitor type-1 (PAI-1). The objective of the present study was to characterize the localization, ontogeny and regulation of PAI-1 expression within ovine corpora lutea. Urokinase binding activity was detected within medium conditioned by ovine luteal cells. Production of PAI-1 by ovine luteal cells was confirmed by immunoprecipitating it from labelled proteins in culture medium. mRNA encoding PAI-1 was present within developing (day 3), mature (day 10) and regressing (30 h after prostaglandin F2α injection on day 10 after the onset of oestrus) corpora lutea as demonstrated by in situ hybridization. The ontogeny of PAI-1 mRNA expression was characterized within corpora lutea collected on days 3, 7, 10, 13 and 16 after the onset of oestrus (n = 4, 4, 4, 3 and 4, respectively). Expression of PAI-1 mRNA did not differ during the luteal phase (P= 0.06), although a trend for an increase in the amount of PAI-1 mRNA was observed on day 16. Expression of PAI-1 mRNA was also examined during luteal regression in corpora lutea collected 0, 6, 12, 24 and 36 h after injection of prostaglandin F2α on day 10 after the onset of oestrus (n = 4 at each time). Relative PAI-1 mRNA concentrations changed significantly during luteolysis induced by prostaglandin F2α (P = 0.0002). Administration of prostaglandin F2α resulted in a transient sevenfold increase in PAI-1 mRNA 6 h after injection (P = 0.0001) but by 12 h the amounts had returned to values similar to those detected on day 10. We conclude that PAI-1 is a major secretory product of ovine luteal cells and that a transient increase in PAI-1 mRNA occurs during luteolysis induced by prostaglandin F2α. PAI-1 probably plays a key local role in the control of extracellular proteolysis during the luteal phase.