Early pregnancy diagnosis in sheep by progesterone and pregnancy-associated glycoprotein tests

The aim of this study was to compare the accuracy of the progesterone (P4) and pregnancy associated glycoprotein (PAG) tests for determination of early pregnancy in sheep. Estrus was synchronized in 182 Awassi x Merino ewes and blood samples were collected at Days 0 (day of the insemination), 18, 22, 29, 36, and 50 after artificial insemination (AI). Plasma P4 concentrations at Days 0 and 18 were determined by double antibody radioimmunoassay, while PAG concentrations at Days 22, 29, 36 and 50 were determined by a heterologous, double-antibody radioimmunoassay (RIA) using the bovine PAG 67 kDa subunit as tracer and standard and rabbit antiserum raised against a mixture of caprine 55 and 59 kDa PAG subunits as the first antibody. The discriminatory value for diagnosis of pregnancy by the P4 and the PAG-RIA tests was > or = 1 ng/ml. Based on lambing data, the accuracy for diagnosing pregnant (sensitivity) and non-pregnant ewes (specificity) and predictivity of both tests were calculated. The sensitivity, specificity, positive and negative predictive values for P4 and PAG tests were 100, 95.4, 81.6, and 100% at Day 18 (P4) and 93.5, 100, 100 and 98.7% at Day 22 (PAG), respectively. For diagnosis of non-pregnant ewes the PAG test had significantly higher specificity than the P4 test (P < 0.05). It is concluded that ovine pregnancy can be reliably diagnosed at Day 22 after AI by using a heterologous radioimmunoassay of PAG.     

Estrogens and progesterone during gestation in Dolichotis patagona

During pregnancy, a progesterone-binding plasma protein is present and is similar in several respects to the binding protein reported in other hystricomorphs. These findings establish estradiol-17 beta as the predominant estrogen of pregnancy and that progesterone rises during pregnancy and does not decline until after parturition. Gestation length is 96 days. This study establishes similarities between the mara and its closest relative, the guinea pig.     

Genetic diversity in the Churra tensina and Churra lebrijana endangered Spanish sheep breeds and relationship with other Churra group breeds and Spanish mouflon

The aim of the present study was to estimate the genetic intra-breed variability of Churra tensina and Churra lebrijana endangered breeds and to establish genetic relationships with Churra, Latxa and Merino breeds, as well as Spanish mouflon, by using 28 microsatellite markers, to provide useful information for their conservation. Allele frequencies and heterozygosity revealed high genetic variation in the two endangered breeds despite their small population size. Estimates of inbreeding coefficient (F_IS) were significant for all breeds studied, except for Churra lebrijana breed. The highest inbreeding coefficient (F_IS = 0.143) was found in the Spanish mouflon. Genetic differentiation tests (F_ST = 0.121) and assignment of individuals to populations indicated the existence of defined breed populations, and low genetic flow between these breeds. The highest pairwise Reynolds distance (D_R) values were observed between Mouflon and the domestic sheep breeds. Considering only domestic sheep breeds, the Churra lebrijana breed showed the highest pairwise D_R values_. The lowest values were found between Latxa and the other domestic sheep, except for Churra lebrijana. Results of pairwise D_R values, as well as phylogenetic tree and bottleneck analysis showed an important genetic isolation of the Churra lebrijana breed from the other Churra types, and genetic signatures of a demographic bottleneck. Finally, structure analysis of populations detected a population subdivision in the Latxa sheep breed. In conclusion, this study presents valuable insight into the existing genetic variability of two Spanish endangered breeds, as well as the first study in Spanish mouflon based on microsatellite analysis. The high degree of variability demonstrated in Churra tensina and Churra lebrijana implies that these populations are rich reservoirs of genetic diversity.     

Altered secretion of pregnancy-associated glycoproteins during gestation in bovine somatic clones

Somatic nuclear transfer (NT) in cattle is often accompanied by severe placental anomalies, hypertrophy, and hydrallantois, which induce a high rate of pregnancy losses throughout gestation. These placental deficits are associated with an abnormal increase of the maternal plasma levels of pregnancy-associated glycoprotein (PAG), produced by the trophoblastic binucleate cells (BNC) of the placenta. The objective of this study was to analyze the origin of the abnormally elevated PAG concentrations in the peripheral circulation of NT recipients during pathological pregnancies. Concentrations of PAG were measured both in maternal blood, in chorionic and cotyledonary tissular extracts from control recipients (after artificial insemination, AI, or in vitro fertilization, IVF) and clone recipients on Day 32, Day 62, and during the third trimester of gestation. Three different radioimmunoassay (RIA) systems were used. One homologous RIA for PSP60, similar to bovine PAG-1 (PAG_67kDa), and two heterologous RIA with PAG_67kDa as standard and tracer, and antisera anti-caprine PAG (AS#706 and AS#708). Circulating and tissular concentrations of bovine placental lactogen (bPL), a glycoprotein also produced by BNC, were determined by RIA at the same stages. The number of BNC in the placental tissues was determined by cell counting after immunostaining with anti PSP60 antibody on tissue sections from control and NT pregnancies. Maternal plasma PAG concentrations were not different among groups on Day 32, but they were significantly higher in NT than in control pregnancies on Day 62 with all three RIA and during the third trimester with two RIA (RIA-PSP60 and RIA with AS#708). Circulating bPL concentrations were undetectable on Days 32 and 62 and were not different in the third trimester between NT and control pregnancies. Tissular amounts of PAG on total proteins were not different between the two groups at all stages studied. No difference was determined in the percentage of PSP60-positive BNC in placental tissues between controls and NT on Day 62 and during the third trimester of pregnancy. Western blots of tissular extracts from placenta showed no major molecular weight changes of PAG in NT pregnancies compared to controls. No differences in maternal circulation concentrations or tissular content of bPL were observed between control and NT pregnancies. In conclusion, the specific increase of PAG in maternal plasma concentrations during abnormal NT pregnancies do not result from a higher proportion of BNC, or an increased protein expression of PAG and could be due to changes in the composition of terminal glycosylation which result into a clearance decrease of PAG from the circulation.     

Peri-pubertal ovulatory events and progesterone profiles of Javanese thin-tail sheep

Pre- and post-pubertal progesterone profiles and ovarian activity of 24 Javanese thin-tail ewe lambs were studied under research-station conditions. The results showed that immaturity of ovarian function before the animals reached puberty was characterized by the occurrence of ovulation without oestrus (66.7%), oestrus without ovulation (12.5%) and failure in some ewe lambs to maintain regular ovarian activity following their first ovulation. A high proportion (39.5%) of corpora lutea was not fully functional. Some pronounced increases in plasma-progesterone concentration were observed before first ovulation, while an ovulation observed by laparoscopy was not always followed by a marked increase in plasma-progesterone concentration. Once the animals reached puberty, the levels and the patterns of change in plasma-progesterone concentration during a cycle or in early pregnancy were similar to those commonly observed in temperate breeds.     

Embryo survival, progesterone profiles and metabolic responses to an increased feeding level during second gestation in sows

This study describes reproductive and metabolic responses in sows fed at two different feeding levels from day 3-35 of second gestation. After insemination, 37 sows were assigned to one of two treatments: 1) Control: 2.5 kg/day of a gestation diet; 2) Plus Feed 3.25 kg/day of a gestation diet (+30%). Sow weight, back fat and loin muscle depth were measured at farrowing, weaning, start of treatment, day 14 after start treatment and end of treatment. Frequent blood samples were taken for progesterone, luteinizing hormone (LH), glucose and insulin, insulin-like-growth-factor-1 (IGF-1), non-esterified-fatty-acids (NEFA) and urea analysis. At day 35 after insemination sows were euthanized and their reproductive tract collected to assess ovarian, embryonic and placental characteristics. Plus Feed sows gained 5.4 kg more weight and 0.9 mm more back fat and tended to be heavier at slaughter compared to Control sows (193 vs. 182 kg, P = 0.06). No difference in loin muscle gain was found. Treatment also did not affect vital embryonic survival, which was 72.1 ± 3.9% for Control and 73.4 ± 3.2% for Plus Feed sows, resulting in, respectively, 15.9 ± 0.9 and 15.7 ± 0.7 vital embryos. No effect of treatment on any of the ovarian, embryonic or placental characteristics was found. Progesterone profiles during the first month of gestation, and LH characteristics at day 14 of gestation were not different between treatments. Progesterone concentration was lower (P < 0.05) 3 h after feeding compared with the prefeeding level on days 7-11 after first progesterone rise for Plus Feed and on days 8-10 after first progesterone rise for Control sows. At day 15, preprandial glucose and insulin concentrations were not different between treatments, insulin peaked later (48 vs. 24 min) and at a higher concentration in Plus Feed than in Control sows. Furthermore, glucose area under the curve (AUC) tended to be lower (−171.7 ± 448.8 vs. 1257.1 ± 578.9 mg/6.2 h, P = 0.06, respectively) for Plus Feed vs. Control sows. IGF-1 concentration was not different between treatments, but NEFA concentrations were lower for Plus Feed vs. Control sows (149.5 ± 9.2 vs. 182.4 ± 11.9 μm/L, respectively, P = 0.04) and urea concentration tended to be higher in Plus Feed than in Control sows (4.3 ± 0.1 vs. 3.9 ± 0.1, respectively, P = 0.13). None of the metabolic parameteres were related to reproductive measures. In conclusion, feeding 30% more feed from day 3 till d 35 of second gestation increased weight gain and resulted in lower NEFA concentrations, but did not affect progesterone, LH or IGF-1 and embryonic and placental characteristics.     

Comparison of accuracy of ultrasonography,progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n = 195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n = 33), December 2006 (n = 92), or January 2007 (n = 70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.     

Comparison of accuracy of ultrasonography, progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n = 195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n = 33), December 2006 (n = 92), or January 2007 (n = 70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.     

Extent and pattern of pregnancy losses and progesterone levels during gestation in Swedish Red and Swedish Holstein dairy cows

Background

Pregnancy loss is a major source of infertility in dairy cows. Despite a fertilization rate after insemination (AI) of approximately 90%, calving rates are 30%–50%, indicating the occurrence of extensive embryonic and foetal losses. The aim of this study was to establish the extent and pattern of embryonic and foetal loss in Swedish Red (SR) and Swedish Holstein (SH) dairy cows, as well as, the relationship to oestrus intensity (OI) and progesterone (P4) concentration. In total, 2130 AIs and 16,176 milk P4 samples from 359 SR and 212 SH dairy cows were included in the study. Pregnancy losses were estimated using data from P4 values combined with AI information and calving data.

Results

Total pregnancy loss from AI to the day of calving was 65%. Early embryonic loss, late embryonic loss and foetal loss were estimated to be 29, 14 and 13%, respectively. There is strong evidence in the literature that P4 concentrations at different time points are associated with pregnancy loss. In the present study, cows with pregnancy losses had significantly higher P4 levels at the day of AI and significantly lower P4 concentration at days 10, 21 and 30 after AI compared to pregnant cows. Swedish Red cows had significantly lower total pregnancy losses compared to SH cows (62% and 68% respectively, P?=?0.017). Early embryonic loss was 6.7% points lower for cows inseminated at a stronger OI (OI?=?3) compared to at a weaker OI (OI?=?2, P?=?0.006). Cows inseminated at ovulation number?≥?5 had significantly lower early pregnancy losses compared to cows inseminated at first or second ovulation (11.5 and 8% points, respectively, P?<?0.05). With an increase of one SD of milk (448 kg ECM) during the first 60 days in milk, early embryonic loss increased by 4.7% points (P?=?0.006).

Conclusions

It is important to increase the number of cows calving per insemination by reducing embryo/foetal loss. This outcome can be achieved by management and breeding for optimal P4 levels at critical time points, and by considering oestrus expression in the breeding programmes to facilitate the correct timing of insemination.

     

Transferrin in fetal sheep cerebrospinal fluid and plasma during gestation

1. Transferrin concentrations in fetal sheep CSF and plasma have been estimated between 31 and 125 days gestation and in the adult, using a radial immunodiffusion assay. 2. The plasma concentration was lowest (183 +/- 35 mg/100 ml) in the earliest fetuses examined (31 days). It increased to over 350 mg/100 ml by 35 days; thereafter it was around the adult value (580 mg/100 ml). 3. In CSF the transferrin concentration increased from 43 +/- 10 mg/100 ml at 31 days to a maximum of 163 +/- 14 mg/100 ml at 40 days gestation after which it decreased considerably to 6.1 +/- 0.7 mg/100 ml at 125 days and was even lower in the adult (1.1 +/- 0.2 mg/100 ml). 4. CSF: plasma ratios for transferrin especially when compared with those of other plasma proteins, are not compatible with passive leakage of protein from blood to CSF in the developing brain. The results may be explained by specific transfer of proteins into CSF but synthesis by the choroid plexus or brain has not been excluded.     

Androgen level in the sheep fetus during gestation.

The androgen content of amniotic fluid, plasma, and gonads from 107 fetal lambs was determined by radioimmunoassay in an attempt to understand the ontogeny of gonadal function. Testosterone (T) was too low to be reliably measured in the amniotic fluid from fetuses of either sex. Ovaries were without T activity at any of the stages of gestation studied. Testicular T-5alpha-dihydrotestosterone (T-DHT) concentration steadily decreased from 1.4/ml near term. It is suggested that nongonadal testosterone production increases during fetal life and that T secretion by the fetal testis may contribute steadily less to the plasma pool of T as gestation proceeds.     

Purification and characterization of a bovine pregnancy-associated glycoprotein

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).     

A comparison of diagnosis of pregnancy in the goat via transrectal ultrasound scanning, progesterone, and pregnancy-associated glycoprotein assays

Real-time ultrasound scanning (US) via the transrectal route, progesterone (P4) assay, and pregnancy-associated glycoprotein (PAG) detection can be used to diagnose pregnancy at around 3 weeks after breeding. Although several studies have been carried out to evaluate each of these different methods individually, it is difficult to establish adequate comparisons due to differences, such as the breed of goat, age, and farming conditions, among others. The aim of the present paper is to compare the accuracy of diagnosis of pregnancy using transrectal US, P4 assay and PAG detection at the same time and on the same animals. Canary dairy goats (n=143) were synchronized with an 11-day fluorogestone acetate (FGA) intravaginal sponge followed by PGF2alpha and eCG 2 days before the FGA withdrawal. Blood samples were collected on Days 20, 22, 24, and 26 after mating to determine P4 and PAG concentrations. Transrectal US examinations were performed at the same time. There were 79 pregnant goats and another 64 non-pregnant. The US via the transrectal method and the determination of PAG concentrations provide very accurate pregnancy diagnosis at 24-26 days after breeding; on the contrary, P4 assay on plasma samples performed on Day 22 after breeding was accurate, in this case, in detecting pregnant animals but did not always detect the non-pregnant does.     

Evolution of tissue levels of steroids: oestradiol-17 beta, oestrone and progesterone in the macaque myometrium during gestation

In 15 cynomolgus monkeys between days 30-160 of gestation, tissue levels of oestrone (E1), oestradiol 17 beta (E2) and progesterone (P4) were assayed by RIA in the myometrium and the placenta. Myometrial samples were subdivided as follows: inner and outer layers adjacent to the placental area (IMP and OMP) and inner and outer layers from antiplacental areas (IMAP and OMAP). Steroid levels (ng/g wet wt) were in the range of known plasma values (ng/ml) and there was no asymmetric distribution of steroids between the various locations. When the results obtained in all the layers were pooled the gestational profiles indicated a decrease of E1 between days 80-130, whereas at the same time E2 and P4 increased. The ratio P4/E2 was 8 on day 40, 17 on day 80 and 9 on day 160. In the placenta, levels of E2 and P4 were 4 times higher, levels of E1 10 times higher than in the myometrium. Gestational profiles of the three steroids in the placenta increased from day 30 to day 160. Myometrium steroid content therefore does not appear to be a simple reflection of steroid diffusion from the site of production.     

Comparison of accuracy of transabdominal ultrasonography, progesterone and pregnancy-associated glycoproteins tests for discrimination between single and multiple pregnancy in sheep

The aim of the present study was to evaluate and, compare the accuracy of transabdominal ultrasonographic (US) and the progesterone (P4-RIA) and ovine pregnancy-associated glycoprotein (ovPAG-RIA) tests for the discrimination between single and multiple pregnancy in sheep. One hundred pregnant AwassixMerino ewes were scanned by transabdominal ultrasonography (3.5 MHz linear-array transducer) at Days 43-56 and 81 of these ewes were scanned at Days 76-87 of gestation. The ewes were scanned in dorsal recumbency at the bare area of the inguinal regions (without pre-scanning shaving of the ventral abdominal wall). After each scan, blood samples were withdrawn from the jugular vein to estimate the levels of P4 and ovPAG by radioimmunoassay. At lambing, 61 ewes gave birth to single lambs and 39 ewes gave birth to multiples. The sensitivity of the transabdominal US, the P4-RIA and the ovPAG-RIA tests for determining ewes carrying multiples was 54, 64.1 and 64.1% at Days 43-56. At Days 76-87 of gestation these accuracies were 60.0, 66.7 and 76.6% for the US, P4-RIA and PAG-RIA tests, respectively. The specificity of the transabdominal US, the P4-RIA and the ovPAG-RIA tests for determining ewes carrying singles, was 78.6, 60.7 and 62.3% at Days 43-56 and 78.4, 64.7 and 70.6% at Days 76-87 of gestation, respectively. It is concluded that the accuracy of transabdominal ultrasonographic (without pre-scanning shaving of the ventral abdominal wall), the P4- and the ovPAG-RIA tests for determination of the fetal numbers in AwassixMerino crossbred ewes is too low to be used in the field.     

Plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations during gestation in Neospora-infected dairy cows

The aim of this study was to determine whether plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations in pregnancy are affected by persistent Neospora caninum infection in dairy cows. The data analyzed were derived from 22 multiparous cows: 16 N. caninum-seropositive and 6 N. caninum-seronegative animals (used as controls). Three of the 16 seropositive cows aborted during the study period and the corresponding data were analyzed separately. Pregnancy diagnoses were performed on day 40 post-insemination by transrectal ultrasound, and by palpation per rectum on days 90, 120, 150, 180 and 210. Blood samples were collected from each animal immediately before each pregnancy diagnosis, and then at parturition or at the time of abortion detection. Plasma was tested for antibodies against N. caninum and PAG-1 concentrations were determined by radioimmunoassay. In non-aborting animals, the effects of neosporosis (seropositive versus seronegative), N. caninum antibody levels, semen providing bull, sex of the newborn, and day of gestation on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance. The effect of the gestation period (first half versus second half) on the N. caninum antibody titer was established by the Student's t-test in seropositive cows. A significant positive effect of gestation day on PAG-1 concentrations was observed (d.f.=6; F=12.6; P<0.0001). For all cows, PAG-1 concentrations increased steadily during the course of gestation, with peak concentrations recorded at parturition. Neosporosis (P=0.493), N. caninum antibody levels (P=0.921), sex of the newborn (P=0.856) and semen providing bull (P=0.087) had no effect on plasma PAG-1 concentration. There was a significant 52% increase (P<0.0001) in N. caninum antibody titers during the second half of gestation compared to the first half. The fates of the three aborting cows were abortion on gestation day 215 in one, and fetus mummification diagnosed on gestation days 180 and 210, respectively, in the remaining two cows. A luteolytic dose of prostaglandin was applied 30 days after mummification diagnosis in these last two cows, and fetus expulsion was detected on days 215 and 250, respectively. Two of the aborted fetuses were submitted to laboratory analysis and the presence of N. caninum was confirmed by specific PCR. In the cows with a mummified fetus, PAG-1 concentrations were low or undetectable when the diagnosis was made. These findings suggest that N. caninum infection has no effect on placental function in chronically infected, cows not suffering abortion, while PAG-1 measurements in aborting animals provide a useful indication of feto-placental status.     

Localization of chorionic pregnancy-associated glycoprotein family in the pig

The objective of this study was to localize the immuno-positive porcine PAG (pPAG) proteins within chorionic cells throughout the intensive placenta development as pregnancy advances (16-61 days post coitum - dpc). Placental sections were used for double fluorescent histochemistry with selected primary rabbit anti-pPAG sera. The polyclonals were created against recombinant pPAG2 antigen or various secretory porcine native chorionic antigens produced in vitro. Among placental cells stained with fluorescent propidium iodine, the positive pPAG immuno-complexes were visualized by Alexa 488 fluorochrom - conjugated to secondary anti-rabbit goat immunoglobulins. This is the first report concerning cellular localization of the pPAG protein family within diffuse epitheliochorial placenta development throughout the first half of pregnancy in the pig. Fluorescent immuno-positive pPAG signals have been restricted to chorionic cell layers (branched mushroom-like and finger-like structures) that generate a epitheliochorial feto-maternal surface augmented by maternal endometrium interdigitations with the gestation progress in the pig. These results suggest that the pPAG proteins robustly expressed in chorionic cells are involved in the regulation of intensive development of diffuse porcine placenta during the first half of pregnancy.     

Regulation of progesterone during follicular development by FSH and LH in sheep

Progesterone (P4) can participate in the development of female mammalian antral follicles through nuclear receptor (PGR). In this experiment, the differences of P4 synthesis and PGR expression in different developmental stages of sheep antral follicles (large > 5mm, medium 2-5mm, small < 2mm) were detected by enzyme-linked immunosorbent assay, immunohistochemistry, qRT-PCR and Western blotting. Secondly, sheep follicular granulosa cells were cultured in vitro. The effects of different concentrations of FSH and LH on P4 synthesis and PGR expression were studied. The results showed that acute steroid regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3β Hydroxysteroid dehydrogenase (3β-HSD) and PGR were expressed in antral follicles, and with the development of antral follicles in sheep, StAR, P450scc and the expression of 3β-HSD and PGR increased significantly. In vitro experiments showed that FSH and LH alone or together treatment could regulate P4 secretion and PGR expression in sheep follicular granulosa cells to varying degrees, hint P4 and PGR by FSH and LH, and LH was the main factor. Our results supplement the effects of FSH and LH on the regulation of P4 synthesis during follicular development, which provides new data for further study of steroid synthesis and function in follicular development.