Problems associated with ultrasonography of the canine ovary around the time of ovulation.

Real-time B-mode ultrasound imaging was carried out, using a scanner of moderate quality, on 40 fresh bitch cadavers to identify the presence of ovaries and significant ovarian structures. The results indicated that accurate identification of the ovaries depended on the presence of significant follicles or corpora lutea (CL). The findings were verified at post-mortem examination. The study was continued by scanning 35 live bitches using three different scanning units of increasingly improved quality to show that the process of ovulation could be assessed with reasonable accuracy with improved quality of equipment, although the exact fate of the follicle could not be imaged. The results were judged against assays for blood circulating progesterone. A final group of seven live bitches were scanned with a high-grade scanner using an annular phased-array transducer to attempt to image the process of ovulation. The non-echogenic follicle disappeared and was replaced by the CL that was hypoechoic with respect to surrounding tissue, thus allowing accurate assessment of the time of ovulation. This was confirmed by progesterone assay.

Acute phase proteins in canine pregnancy (Canis familiaris).

An acute phase response has been identified during mid-gestation in bitches by determination of the serum C-reactive protein (CRP) concentration. In four pregnant bitches, a large increase in CRP during mid-gestation was followed by a second increase after parturition. In three pregnant bitches, a similar increase was observed during mid-gestation but the second (post parturition) increase was not detected. In two pregnant bitches only a small rise was observed in mid-gestation. No increase in CRP was observed in the serum of two bitches that were not mated following oestrus or in the serum of two bitches that were mated but did not become pregnant. One bitch was diagnosed as pregnant by palpation but did not produce a litter and did not have a mid-gestation increase in CRP. However, this animal did show an acute phase response shortly after oestrus.

Molecular biological methods for monitoring oocyte maturation and in vitro fertilization in bitches.

Oocytes were collected from ovaries of bitches, at various stages of the oestrous cycle, after routine sterilization. Cumulus-enclosed oocytes were cultured for 0-72 h in a modified M-199 medium containing 10% oestrous bitch serum and 20 micrograms oestradiol ml-1. For oocytes surrounded by two or more layers of cumulus cells, an increase in the expression of mRNA transcripts for zona pellucida glycoprotein 3 (ZP3) was seen and reached peak levels after 48 h culture in vitro. Histological assessment showed that 39% of these oocytes had extruded their first polar body after 24 h culture in vitro. When these in vitro matured oocytes were transferred to oviduct cell monolayers and inseminated with fresh dog spermatozoa in Medium-199 supplemented with 10% fetal calf serum and 50 mg gentamicin sulfate ml-1, penetration of the zona pellucida started 1 h after insemination for oocytes that had been cultured for 48 and 72 h. At 12 h after insemination both male and female pronuclei were seen in 37.5% and 20% of the oocytes incubated for 48 and 72 h, respectively. No further development was seen.

Comparison of endocrine changes and ultrasound as means of identifying ovulation in the bitch.

Twenty bitches were monitored through pro-oestrus and oestrus using both circulating plasma hormone levels and ultrasonic examination of the ovaries. Using luteinising hormone (LH) as being the generally accepted optimum indicator of the day of ovulation, comparisons were made of the accuracy of progesterone and ultrasound to identify ovulation. Progesterone agreed with LH in 12 of 20 bitches and was within one day in seven of the other eight. Ultrasound was less accurate in that only four of the 16 estimates agreed, with a further six being within one day. However, if only the bitches which were examined by ultrasound with the latest equipment were included, while only three of 11 coincided, six of the remaining eight were within one day of the LH estimated ovulation. It is concluded that, at present, of the rapid assessments, the measurement of plasma progesterone is a better estimator of ovulation than ultrasound.

Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase.

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.

Detection of the mRNA to placental alkaline phosphatase by in vitro translation and immunoprecipitation.

RNA prepared from human placental syncytiotrophoblast tissue was analysed by in vitro translation and immunoprecipitation. The major polypeptide synthesized from syncytiotrophoblast mRNA was human placental lactogen, accounting for 15-20 per cent of the translated polypeptides. Immunoprecipitation using antibodies to placental alkaline phosphatase (PLAP) specifically precipitated polypeptides of 56 kD and 58 kD. The possible relationship between these polypeptides and those of placental microvillus PLAP is discussed.

A search for antibodies in term maternal sera to solubilized syncytiotrophoblast surface components. A passive haemagglutination assay yields negative evidence.

Using a passive haemagglutination assay, agglutinins against trophoblast components were detected in term maternal sera at titres above those of non-pregnant control sera. These agglutinin titres were independent of the family relationship between individual sera and trophoblast samples. They were unaffected by adsorption with excess packed fetal or paternal red blood cells or paternal lymphocytes. The agglutinins of both normal control and term maternal sera were found to elute in the macroglobulin fraction of serum having a molecular weight of greater than 350 kDa and were shown to be a component of euglobulin precipitates. The titres of the agglutinins were unaffected by adsorption using solid phase antibodies against human IgM and IgG from the sera. It was concluded that these agglutinins were not a maternal antibody response against trophoblast surface components.