Effect of retinol in the vitrification medium on viability of vitrified ovine preantral follicles and expression of key developmental and apoptosis related genes.

BACKGROUND: Vitrification increases the production of reactive oxygen species (ROS) and the antioxidants in the vitrification solution may be beneficial by reducing excessive ROS production. OBJECTIVE: To evaluate the effect of retinol supplementation in vitrification solution on viability, apoptosis and development-related gene expression in vitrified sheep preantral follicles. MATERIALS AND METHODS: Preantral follicles were isolated and randomly assigned into one of five groups: Group1, control fresh preantral follicles; Group 2, vitrification treatment; Group 3, vitrification + 2 µM retinol; Group 4, vitrification + 5 µM retinol; Group 5, vitrification + 10 µM retinol. Preantral follicles were placed in vitrification solutions and then plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue exclusion method and for gene expression. RESULTS: Vitrification with 5 µM retinol positively affected viability in comparison with vitrification without retinol (P < 0.05). There was no significant difference in viability among the Group 1, Group 2, Group 3 and Group 5. Expression of apoptotic genes BAX and Casp 3 were higher in the vitrified group, and vitrification with 5 µM retinol (Group 4) is comparable to the control fresh. Expressions of other apoptosis-related genes (i.e., BCL2L1, BAD and BAK) showed significant difference between the control fresh group and the vitrification group with 5 µM retinol. Expression of Annexin5 was also significantly different among various groups. The expression of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in the Group vitrified with 5 µM retinol. CONCLUSION: The supplementation of 5 µM retinol in vitrification solution was beneficial for the vitrification of ovine preantral follicles.

Effect of different vitrification protocols on post thaw viability and gene expression of ovine preantral follicles.

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200-300 µm) were randomly assigned into four groups. Group1 - Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2- FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2- FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (-196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

Effect of retinol as antioxidant on the post-thaw viability and the expression of apoptosis and developmental competence-related genes of vitrified preantral follicles in buffalo (Bubalus bubalis).

The present study evaluated the effect of supplementation of retinol in the vitrification solution on the viability, apoptosis and development-related gene expression in vitrified buffalo preantral follicles. Preantral follicles isolated from cortical slices of ovaries were randomly assigned into three groups: Group1-Control fresh preantral follicles; Group 2-Vitrification treatment (Vitrification solution 1 (VS1) -TCM-199 + 25 mM HEPES + Foetal bovine serum (FBS) 10%, Ethylene glycol (EG): 10%, Dimethyl sulphoxide (DMSO): 10%, Sucrose-0.3 M for 4 min; VS2- TCM-199 + 25 mM HEPES + FBS10%, EG:25%, DMSO: 25%, Sucrose:0.3 M for 45 s); Group3-vitrification treatment +5 µM of Retinol. Preantral follicles were placed in corresponding vitrification medium and plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analysed for follicular viability and gene expression. There was no significant difference in the viability rates among the Group 1(Fresh preantral follicles) (91.46 ± 2.39%), Group 2 (89.59 ± 2.46%) and Group 3 (87.19 ± 4.05%). There was a significantly (p < .05) higher mRNA expression of BCL2L1, GDF-9 and BMP-15 in the vitrification + retinol group compared with the control group. There was a significantly (p < .05) higher expression of Caspase-3 and Annexin-5 in the vitrification group and Vitrification + retinol group compared with control group of follicles. It is concluded that the supplementation of 5 µM of Retinol in Vitrification solution was an efficient vitrification procedure for the vitrification of buffalo preantral follicles.

Disturbed spermatogenic signaling in pituitary adenylate cyclase activating polypeptide-deficient mice.

PACAP is a neuropeptide with diverse functions in various organs, including reproductive system. It is present in the testis in high concentrations, and in addition to the stage-specific expression within the seminiferous tubules, PACAP affects spermatogenesis and the functions of Leydig and Sertoli cells. Mice lacking endogenous PACAP show reduced fertility, but the possibility of abnormalities in spermatogenic signaling has not yet been investigated. Therefore, we performed a detailed morphological analysis of spermatozoa, sperm motility and investigated signaling pathways that play a role during spermatogenesis in knockout mice. No significant alterations were found in testicular morphology or motility of sperm in homozygous and heterozygous PACAP-deficient mice in spite of the moderately increased number of severely damaged sperms. However, we found robust changes in mRNA and/or protein expression of several factors that play an important role in spermatogenesis. Protein kinase A expression was markedly reduced, while downstream phospho-ERK and p38 were elevated in knockout animals. Expression of major transcription factors, such as Sox9 and phospho-Sox9, was decreased, while that of Sox10, as a redundant factor, was increased in PACAP-deficient mice. The reduced phospho-Sox9 expression was partly due to increased expression and activity of phosphatase PP2A in knockout mice. Targets of Sox transcription factors, such as collagen type IV, were reduced in knockout mice. In summary, our results show that lack of PACAP leads to disturbed signaling in spermatogenesis, which could be a factor responsible for reduced fertility in PACAP knockout mice, and further support the role of PACAP in reproduction.

Relationship between Paratuberculosis and the microelements Copper, Zinc, Iron, Selenium and Molybdenum in Beef Cattle.

To study the deficiency of minerals and its relationship with Paratuberculosis, blood, serum, and fecal samples were obtained from 75 adult bovines without clinical symptoms of the disease and from two bovines with clinical symptoms of the disease, from two beef herds with a previous history of Paratuberculosis in the Province of Buenos Aires, Argentina. Serum samples were processed by ELISA and feces were cultured in Herrolds medium. Copper, zinc and iron in serum were quantified by spectrophotometry and selenium was measured by the activity of glutathione peroxidase. We also determined copper, zinc, iron and molybdenum concentrations in pastures and the concentration of sulfate in water. Mycobacterium avium subsp paratuberculosis (Map) was isolated from 17.3% of fecal samples of asymptomatic animals and from the fecal samples from the two animals with clinical symptoms. All the Map-positive animals were also ELISA-positive or suspect, and among them, 84.6% presented low or marginal values of selenium and 69.2% presented low or marginal values of copper. The two animals with clinical symptoms, and isolation of Map from feces and organs were selenium-deficient and had the lowest activity of glutathione peroxidase of all the animals from both herds. All the animals negative to Map in feces and negative to ELISA had normal values of Se, while 13.8% of animals with positive ELISA or suspect and culture negative presented low levels of Se. Half of the animals that were negative both for ELISA and culture in feces were deficient in copper but none of them presented low values of selenium. The content of molybdenum and iron in pasture was high, 2.5 ppm and 1.13 ppm in one herd and 2.5 ppm and 2.02 ppm in the other, respectively, whereas the copper:molybdenum ratio was 1.5 and 5.2, respectively. These results do not confirm an interaction between imbalances of the micronutrients and clinical Paratuberculosis, but show evidence of the relationship between selenium deficiencies in animals with Map infection and ELISA positive results.

Relationship between Paratuberculosis and the microelements Copper, Zinc, Iron, Selenium and Molybdenum in Beef Cattle

To study the deficiency of minerals and its relationship with Paratuberculosis, blood, serum, and fecal samples were obtained from 75 adult bovines without clinical symptoms of the disease and from two bovines with clinical symptoms of the disease, from two beef herds with a previous history of Paratuberculosis in the Province of Buenos Aires, Argentina. Serum samples were processed by ELISA and feces were cultured in Herrolds medium. Copper, zinc and iron in serum were quantified by spectrophotometry and selenium was measured by the activity of glutathione peroxidase. We also determined copper, zinc, iron and molybdenum concentrations in pastures and the concentration of sulfate in water. Mycobacterium avium subsp paratuberculosis (Map) was isolated from 17.3% of fecal samples of asymptomatic animals and from the fecal samples from the two animals with clinical symptoms. All the Map-positive animals were also ELISA-positive or suspect, and among them, 84.6% presented low or marginal values of selenium and 69.2% presented low or marginal values of copper. The two animals with clinical symptoms, and isolation of Map from feces and organs were selenium-deficient and had the lowest activity of glutathione peroxidase of all the animals from both herds. All the animals negative to Map in feces and negative to ELISA had normal values of Se, while 13.8% of animals with positive ELISA or suspect and culture negative presented low levels of Se. Half of the animals that were negative both for ELISA and culture in feces were deficient in copper but none of them presented low values of selenium. The content of molybdenum and iron in pasture was high, 2.5 ppm and 1.13 ppm in one herd and 2.5 ppm and 2.02 ppm in the other, respectively, whereas the copper:molybdenum ratio was 1.5 and 5.2, respectively. These results do not confirm an interaction between imbalances of the micronutrients and clinical Paratuberculosis, but show evidence of the relationship between selenium...

Assisted reproductive technologies in the reproductive management of small ruminants.

In modern agriculture, assisted reproductive technologies are being used for out of season oestrus induction, enhancement of reproductive performance and genetic improvement. In addition, they can have substantial contribution in preservation of endangered species or breeds, as well as in eradication programs of various diseases. While their applications are widespread in cattle, in small ruminants it is almost restricted to artificial insemination. The main limitations of a wider application in small ruminants are the naturally occurring anoestrus period, the variability of response to superovulatory treatments, the fertilisation failure and the need of surgery for collection and transfer of gametes and embryos. Nonetheless, during the last 30 years, considerable progress has been made in sheep and goat embryo technologies, especially in the fields of oestrus synchronisation, superovulation and in vitro embryo production. This paper reviews the status of assisted reproductive technologies in sheep, analysing the prospects offered by recent advances in in vivo and in vitro embryo production from mature and juvenile lambs.

Semen processing and artificial insemination in health management of small ruminants.

Artificial insemination has changed the small ruminant industry and has allowed increased genetic improvement, better control of reproduction and sexually transmitted diseases, dissemination of valuable genetics and preservation of the genetics of endangered breeds. Recent developments in this technology have focused on preserving the vitality/fertilizing capability of fresh and frozen spermatozoa by improving the composition of extenders, and by changing cooling/freezing protocols. The other main issue is the development of minimal invasive techniques for proper deposition of fresh or frozen semen. The paper discusses state of the art in methodology and technology currently used in small ruminant artificial insemination, as well as future perspectives after their wide application in these animal species.

Comparison of three methods for gastrointestinal nematode diagnosis determination in grazing dairy cattle in relation to milk production.

Development of resistance to anthelmintic drugs has motivated the search for diagnostic methods to identify animals for targeted selective treatments. We compared three methods for the diagnosis of nematode infection in relation to milk production in a fully grazing dairy herd of 150 cows in the humid Pampa (Argentina). Animals had feces, blood and milk sampled during the first postpartum month for EPG, pepsinogen and anti-Ostertagia antibody determination, respectively. With the results obtained two groups of cows, divided in high and low parasite burden, were conformed for each method, and milk production was then compared between groups. When cows were separated by the EPG method (EPG=0 (N=106) vs. EPG>0 (N=44)) a difference of nearly 800 l of milk per cow per lactation was found (P<0.05). On the other hand, milk production between groups separated by Pepsinogen (mUtyr ≤ 1000 vs. mUtyr > 1000) or by anti-Ostertagia (ODR ≤ 0.5 vs. ODR > 0.5) results did not differ. Interestingly, proportion of cows in each group differed between methods (P<0.0001), and the anti-Ostertagia method yielded significantly more cows in the high index group compared to results using the EPG or Pepsinogen method. No correlations were found between parasite indexes determined by the different methods. High parasite burden estimation found may be ascribed to the production system, fully grazing all year round, and to the sampling time, at the beginning of lactation with cows in negative energy balance and depressed immunity. The fact that the cows were born and reared outside, on pasture with continuous nematode larvae exposure, may also account for the results obtained. In conclusion, EPG counting during the first postpartum month may be a useful tool for the diagnosis of production impairment induced by high nematode burden in adult grazing dairy cows. The anthelmintic treatment of only the EPG-positive recently calved cows would improve milk production, while reducing selective pressure on nematode population for the development of resistance.

Subsequent effect of subacute T-2 toxicosis on spermatozoa, seminal plasma and testosterone production in rabbits.

Pannon White (n=12) male rabbits (weight: 4050 to 4500 g, age: 9 months) received 2 ml of a suspension containing purified T-2 toxin by gavage for 3 days. The daily toxin intake was 4 mg/animal (0.78 to 0.99 mg/kg body weight (BW)). Control animals (n=12) received toxin-free suspension for 3 days. Since a feed-refusal effect was observed on the second day after T-2 administration, a group of bucks (n=10) were kept as controls (no toxin treatment) but on a restricted feeding schedule, that is, the same amount of feed was provided to them as was consumed by the exposed animals. On day 51 of the experiment (i.e. 48 days after the 3-day toxin treatment), semen was collected, and pH, concentration, motility and morphology of the spermatozoa, as well as concentration of citric acid, zinc and fructose in the seminal plasma, were measured. After gonadotropin-releasing hormone (GnRH) analogue treatment, the testosterone level was examined. One day of T-2 toxin treatment dramatically decreased voluntary feed intake (by 27% compared to control, P<0.05) and remained lower (P<0.05) during the first 2 weeks after the withdrawal of the toxin. BW of the contaminated rabbits decreased by 88% on days 17 and 29 compared to controls (P<0.05). No effect of toxin treatment was detected on pH and quantity of the semen or concentration of spermatozoa. The ratio of spermatozoa showing progressive forward motility decreased from 65% to 53% in the semen samples of toxin-treated animals compared to controls (P>0.05). The ratio of spermatozoa with abnormal morphology increased (P<0.05) in the ejaculates collected from the toxin-treated animals. T-2 toxin applied in high doses decreased the concentration of citric acid in seminal plasma (P<0.05). No effect of T-2 toxin on the concentrations of the other seminal plasma parameters (fructose and zinc) was observed. T-2 toxin decreased the basic testosterone level by 45% compared to control (P<0.01) and resulted in lower (P<0.05) GnRH-induced testosterone concentration. Feed restriction, that is, less nutrient intake, resulted in more morphologically abnormal spermatozoa in the semen, but it did not cause significant loss in BW, motility of the spermatozoa, composition of the seminal plasma or testosterone concentration--its effect needs further examination.

Matrix metalloproteinase activity assays: Importance of zymography.

INTRODUCTION: Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases capable of degrading extracellular matrix, including the basement membrane. MMPs are associated with various physiological processes such as morphogenesis, angiogenesis, and tissue repair. Moreover, due to the novel non-matrix related intra- and extracellular targets of MMPs, dysregulation of MMP activity has been implicated in a number of acute and chronic pathological processes, such as arthritis, acute myocardial infarction, chronic heart failure, chronic obstructive pulmonary disease, inflammation, and cancer metastasis. MMPs are considered as viable drug targets in the therapy of the above diseases. METHODS: For the development of selective MMP inhibitor molecules, reliable methods are necessary for target validation and lead development. Here, we discuss the major methods used for MMP assays, focusing on substrate zymography. We highlight some problems frequently encountered during sample preparations, electrophoresis, and data analysis of zymograms. RESULTS AND DISCUSSION: Zymography is a widely used technique to study extracellular matrix-degrading enzymes, such as MMPs, from tissue extracts, cell cultures, serum or urine. This simple and sensitive technique identifies MMPs by the degradation of their substrate and by their molecular weight and therefore helps to understand the widespread role of MMPs in different pathologies and cellular pathways.

The effect of long-term in vivo culture in bovine oviduct and uterus on the development and cryo-tolerance of in vitro produced bovine embryos.

The objective of this study was to compare the embryo production and quality carried out entirely in vitro or partly in vitro combined with short- vs long-term in vivo culture using the homologous cattle oviduct. The IVM oocytes were in vitro fertilized and cultured for 7 and 8 days (IVP-Group), or after IVF and 2-3 days of IVC, 4-8 cell stage embryos were endoscopically transferred into oviducts of synchronized heifers (In Vivo-Group) or IVM oocytes were co-incubated with spermatozoa for 3-4 h and transferred into the oviducts of synchronized heifers (GIFT-Group). Embryos of the In Vivo-Group and the GIFT-Group were recovered on day 7 from the oviducts and uterine horns. Embryos of all groups were either cryopreserved at day 7 (day 7 blastocysts) or cultured in vitro in CR1aa-medium supplemented with 5% ECS for further 24 h and cryopreserved (day 8 blastocysts). The total blastocyst yield found in the in vivo cultured groups was similar to the results of the IVP-Group. But the appearance of blastocysts was dependent on the duration of in vivo culture. The more time the embryos spent in the in vivo environment, the more blastocysts appeared at day 8. The quality of produced blastocysts assessed by cryo-survival was also correlated to the culture conditions; the in vivo cultured embryos showed higher cryo-tolerance. However, the duration of in vivo culture crucially influenced the cryo-tolerance of produced blastocysts. It is concluded that tubal access is a promising tool to provide a further basis for studying embryo sensitivity to environmental changes.

Endocrine characteristics of late pregnant hyperketonaemic ewes and their reproductive performance following the induction of ovarian cyclicity out of the breeding season.

Ketosis was diagnosed in a flock of Merino ewes that conceived from synchronised oestrus in the early autumn period. On day 140 of pregnancy the ewes were sampled for determination of betaOH-butyrate (BHB), AST, glucose, non-esterified fatty acids (NEFA), total cholesterol (TCH), insulin, T4, T3, cortisol, IGF-1 and leptin. The results were evaluated according to the number of fetuses born some days later and the presence of hyperketonaemia (BHB: > or = 1.60 mmol/l). In May, about 3 months after lambing, cyclic ovarian function was induced (Cronolone + eCG), and the ewes were inseminated artificially (AI) 48 h after the removal of gestagen-containing sponge. At the time of AI and 10 days later blood samples were collected again to check the plasma levels of the same constituents as previously (in samples taken at AI), and to monitor the ovarian response by assaying progesterone (in both samples). On day 140 of gestation significantly lower BHB levels were detected in dams with single (n = 41) than in those with twin (n = 57) pregnancies. Hyperketonaemia was found only in ewes bearing twins (n = 27). These animals had higher NEFA and cortisol, and lower TCH, insulin, IGF-1, leptin and T3 levels than their normoketonaemic twin-bearing flock-mates, and those with single pregnancy. The blood glucose concentrations varied within a wide range, and the means of groups did not exhibit any significant differences. The formerly hyperketonaemic individuals were characterised by lower leptin level 3 months after lambing, and they showed a poorer response to the cycle-induction procedure than the others. The non-responders had lower IGF-1 and leptin levels than those ovulated after this treatment. It was concluded that the subclinical form of ovine ketosis is characterised by complex endocrine alterations, reflecting an obvious form of negative energy balance. If attempts to induce cyclic ovarian function outside the breeding season are made soon after lambing, the ovarian response and fertility of these ewes may also be depressed.

Survival of mouse blastocysts after low-temperature preservation under high pressure.

Cryoinjuries are almost inevitable during the freezing of embryos. The present study examines the possibility of using high hydrostatic pressure to reduce substantially the freezing point of the embryo-holding solution, in order to preserve embryos at subzero temperatures, thus avoiding all the disadvantages of freezing. The pressure of 210 MPa lowers the phase transition temperature of water to -21 degrees C. According to the results of this study, embryos can survive in high hydrostatic pressure environment at room temperature; the time embryos spend under pressure without significant loss in their survival could be lengthened by gradual decompression. Pressurisation at 0 degrees C significantly reduced the survival capacity of the embryos; gradual decompression had no beneficial effect on survival at that stage. Based on the findings, the use of the phenomena is not applicable in this form, since pressure and low temperature together proved to be lethal to the embryos in these experiments. The application of hydrostatic pressure in embryo cryopreservation requires more detailed research, although the experience gained in this study can be applied usefully in different circumstances.

Assisted reproductive research: laser assisted hatching and spindle detection (spindle view technique).

Animal experiments are very important for the development of new assisted reproductive techniques (ART) for use in human and animal reproductive medicine. Most technical aspects of reproductive manipulation of humans and animals are very similar, and many components of successful human ART used nowadays have been derived from animal studies. In this study we examined (1) the use of 'non-contact' laser for assisted hatching, (2) whether spindles in living mouse oocytes could safely be imaged/examined by polarisation microscope (polscope) and (3) the influence of environment (e.g. temperature, in vitro culture, etc.) on spindle detection/visualisation. The data of the study presented here show that (1) laser assisted hatching (AH) is a fast, very accurate and safe procedure without any harmful effect on embryo development and it can support very effectively the implantation of embryos, (2) the use of polscope facilitates the evaluation of oocyte quality and the selection of oocytes with spindle, (3) by monitoring the spindle position during intracytoplasmic sperm injection (ICSI), we can reduce spindle damage and increase the chance of fertilisation. Further studies are underway to test the hypothesised connection between spindle birefringence and developmental capacity of oocytes/embryos.

Effect of the El Niño phenomenon on the ovarian responsiveness and embryo production of donor cows.

The effects of different Temperature Humidity Index (THI) values in cold, hot and El Niño (EN) climates on superovulation and embryo production were analysed on Holstein Friesian donor cows. There were significant differences in the THI among the three climates. The average temperature in the EN period was 6 degrees C higher than in the summer period of the previous 30 years. The number of corpora lutea (CL) and embryos were log- and back-transformed, Kolmogorov-Smirnoff test was used for normality and Lilliefors test was applied for significance. In the cold season THI was 70.74 +/- 1.35 and the average number of CL was 9.84 +/- 4.37. In the hot season the THI was 73.99 +/- 0.72 and the average number of CL was 9.70 +/- 4.49. When the THI, in the EN period, increased up to 79.74 +/- 4.01, the superovulation response was significantly (P < 0.01) reduced (average number of CL = 5.22 +/- 2.53). The embryo production result showed a similar tendency. In the hot period the average number of embryos obtained was 5.87 +/- 2.98. However, in the EN period it decreased to 4.21 +/- 2.05. Higher temperature reduced embryo quality. The proportion of live embryos (%) was 59.2 +/- 37.4 in the cold and 38.2 +/- 38.5 in the EN periods of the year (P < 0.01). However, ovarian sensitiveness showed adaptation to summer environment while the heat stress, which was more severe in the EN period, negatively affected the superovulation response and embryo production.

Superovulation using recombinant human FSH and ultrasound-guided transabdominal follicular aspiration in baboon (Papio anubis).

The response of baboon females to a modified human ovarian stimulation protocol incorporating start of pituitary suppression in the luteal phase of the cycle with a GnRH agonist (GnRHa) and recombinant human FSH (rhFSH) was studied. A long-acting GnRHa implant supplying goserelin acetate was administered s.c. to six adult female baboons experiencing regular menstrual cycles (33-34 days) on days 22-24 of the cycle. Follicular development was monitored by transabdominal ultrasonography and serum levels of E2 and progesterone (P4) and rhFSH were determined by ELISA. Menses occurred 9-10 days after GnRHa administration. Daily i.m. administration of 75 IU rhFSH commenced 9-10 days after menses and continued for 9-10 days. When most follicles were > or =5mm diameter and serum E2 had reached its maximum level, 2000 IU hCG was administered i.m. to induce follicle maturation. Transabdominal ultrasound-guided follicular aspiration of follicles > or =2mm diameter was performed 30-34h after hCG administration. One baboon did not show an adequate response to rhFSH stimulation. This animal did not receive further treatment and no data for it are presented. The number of follicles aspirated was 21+/-4 and 17.2+/-3.8 oocytes were recovered per animal with an average recovery rate of 82% (86/105). The number of oocytes collected from five animals were 14, 21, 16, 15, and 20 (n=86). Most of the oocytes recovered were in metaphase II and 3h after recovery 91% (78/86) were considered suitable for in vitro fertilization. It was concluded that recombinant human FSH can successfully induce follicular recruitment and oocyte maturation in baboon females during pituitary suppression with a GnRHa

Substrate specificities of recombinant mannan-binding lectin-associated serine proteases-1 and -2.

Mannan-binding lectin (MBL)-associated serine proteases-1 and 2 (MASP-1 and MASP-2) are homologous modular proteases that each interact with MBL, an oligomeric serum lectin involved in innate immunity. To precisely determine their substrate specificity, human MASP-1 and MASP-2, and fragments from their catalytic regions were expressed using a baculovirus/insect cells system. Recombinant MASP-2 displayed a rather wide, C1s-like esterolytic activity, and specifically cleaved complement proteins C2 and C4, with relative efficiencies 3- and 23-fold higher, respectively, than human C1s. MASP-2 also showed very weak C3 cleaving activity. Recombinant MASP-1 had a lower and more restricted esterolytic activity. It showed marginal activity toward C2 and C3, and no activity on C4. The enzymic activity of both MASP-1 and MASP-2 was specifically titrated by C1 inhibitor, and abolished at a 1:1 C1 inhibitor:protease ratio. Taken together with previous findings, these and other data strongly support the hypothesis that MASP-2 is the protease that, in association with MBL, triggers complement activation via the MBL pathway, through combined self-activation and proteolytic properties devoted to C1r and C1s in the C1 complex. In view of the very low activity of MASP-1 on C3 and C2, our data raise questions about the implication of this protease in complement activation.

Interaction properties of human mannan-binding lectin (MBL)-associated serine proteases-1 and -2, MBL-associated protein 19, and MBL.

The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8-3.2 S) in the presence of Ca(2+). Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K:(D) values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca(2+)-dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca(2+)-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.

Controlled ovarian stimulation and ultrasound guided follicular aspiration in the baboon (Papio cynocephalus anubis).

The objective of this study was to investigate whether baboon females respond to an ovarian stimulation protocol incorporating pituitary suppression with a GnRH agonist (GnRHa) and highly purified human FSH (hphFSH) with follicular development and oocyte maturation. An ovulation induction protocol was applied to 5 adult female baboons with a history of regular menstrual cycles (33-34 days). A long-acting GnRHa implant containing goserelin acetate was placed s.c. on days 22-24 of their menstrual cycle. Daily hphFSH (75 IU im) treatments were started approximately 10 days following menses. When the majority of the follicles were > or = 5 mm in diameter and the E2 levels had reached a maximum, hCG (2000 IU i.m.) was administered to induce final maturation of the oocytes and ovulation. 30 to 34 h after hCG administration, transabdominal follicular aspiration was performed using a variable frequency transvaginal transducer with ultrasound. A total of 71 oocytes were collected (average: 17). 91% of the oocytes were morphologically normal indicating that they were appropriate for in vitro insemination.