Blood vessel expansion and reduction in the corpus luteum (CL) is regulated by the vascular endothelial growth factor (VEGF) system and linked to the maintenance of the CL. The VEGF system has both angiogenic and antiangiogenic ligands and receptors. Our objective was to evaluate the relationship between the mRNA expression of angiogenic and antiangiogenic members of the VEGF system in the CL, throughout the luteal phase of the oestrous cycle in cows. The CL of 18 cows were collected by transvaginal surgery on days 4, 6, 9, 12, 15 and 18 of the oestrous cycle and the mRNA expression of VEGF system components was evaluated by quantitative real-time PCR. The mRNA expression of VEGF ligands and receptors increased (P<0.05) from the early- and mid-luteal phase (days 4 to 12) reaching its maximum expression on day 15 of the cycle. We found no expression of VEGF164b throughout the cycle. Expression of sVEGFR1 did not change during the oestrous cycle and exceeded that of the VEGFR1 by 100 times. Nonetheless, as VEGFR1 increased, the relationship between the soluble and membrane receptor decreased (P<0.01). In contrast, the expression of VEGFR2 was higher than that of its soluble isoform for all days studied, however, the ratio between the membrane-bound and its soluble counterpart decreased continuously throughout the cycle (P<0.01). Our results show that the expression levels for VEGF ligands, receptors and their antagonistic counterparts are adjusted during CL development and regression, to upregulate angiogenesis early in the oestrous cycle and restrict it at the time of luteolysis.

Current ultrasound techniques can accurately determine the chorionicity of twins, but not zygosity. We previously proposed that the zygosity of spontaneously conceived twins can be determined at early ultrasound, where 2 corpora lutea infers dizygosity, and 1 implies monozygosity. Here we did a case series, comparing zygosity predicted using this method with definitive DNA genotyping of twins after birth. We retrospectively identified 14 ultrasound reports of spontaneous twin pregnancies at 6(+0 days) to 13+6 weeks' gestation, where both ovaries were seen and the number of corpora lutea documented. We visited all twin pairs, obtained buccal smears, and determined zygosity by genotyping 9 independent microsatellite markers. All 8 cases where 2 corpora lutea were seen were dizygotic pregnancies. One further case where 3 corpora lutea were seen was also dizygotic. All 3 sets of monozygotic twins had 1 corpus luteum. There were 2 cases incorrectly assigned, where 1 corpus luteum was seen in dizygotic pregnancies. We conclude if 2 corpora lutea are seen at a first trimester ultrasound of spontaneously conceived dichorionic twins, they appear to be almost certainly dizygotic. However, if 1 corpus luteum is seen in dichorionic twins, zygosity cannot be determined with certainty since it is either monozygotic, or dizygotic where a second corpus luteum has been missed.

The present study examines the size distribution of ovine steroidogenic and non-steroidogenic luteal cells throughout pregnancy. Cells were isolated from corpora lutea collected from early (< 8 weeks), mid (9 to 14 weeks) or late (15 to 18 weeks) stages of pregnancy. Cells were stained for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity, a marker for steroidogenic cells. Both 3β-HSD positive and β-HSD negative cells covered a wide spectrum of size ranging from 7 to 37 μm in diameter. There was a significant increase (P > 0·01) in mean diameter of non-steroidogenic luteal cells as pregnancy progressed. Mean diameter of 3β-HSD negative cells increased from 17·8 (s.e. 0·4) μm in the corpus luteum of early stage of pregnancy to 22·4 (s.e. 0·3) μm in the corpus luteum of advanced pregnancy. However, there was no significant increase in the mean diameter of 3β-HSD positive cells. Corpora lutea obtained from early stages of the pregnancy contained more steroidogenic cells than the cells obtained from mid and late pregnancy (P < 0·01). Percentage of 3β-HSD negative cells had increased 2·07-fold by 18 weeks of pregnancy when compared with the early stage of pregnancy. In contrast, percentage of 3β-HSD positive cells had decreased to 50% of starting values during the same period (P < 0·05). These results indicate that the ovine corpus luteum of pregnancy is morphologically dynamic over the course of pregnancy. Steroidogenic activity of luteal cells may decrease as pregnancy progresses, especially activity of the large luteal cells.

Human ovarian granulosa cells were cultured on a basement membrane preparation (Matrigel) to investigate the role of extracellular matrix components in granulosa cell cluster formation. Time-lapse videomicroscopy of these cultures revealed a rapid aggregation of cells which was initiated during the first 2–4 h of culture so that by 8 h most of the granulosa cells were incorporated into clusters. Further amalgamation then occurred with the transfer of cells along ‘bridges’ between combining clusters. The clustering process, which was complete by about 24 h, was accompanied by reorganisation of matrix which was visualised by immunolabelling of laminin. Clustering cells appeared to gather matrix which became distributed around the clusters. Confocal microscopy showed matrix to be present over the surface of each cluster as well as around the base apparently anchoring the aggregate to the culture surface. Results suggest the potential for active rearrangement of matrix by granulosa-derived cells during corpus luteum development.

The effect of a dietary supplement of calcium soaps of fatty acids on luteal steroidogenic activity was studied in sheep. Mature 3- to 5-year-old North Country Cheviot ewes were given either a pelleted control diet (C; grass, beet pulp, barley; no. = 15) providing daily, 29⋅8 g nitrogen and 13⋅7 MJ of metabolizable energy (ME) or the same diet with 5% (w/w) of the ingredients replaced with protected lipid (C + CaFA; calcium soaps of fatty acids, Megalac, Volac Etd; no. = 15) for 21 days. The latter diet provided daily 30⋅2 g nitrogen and 15⋅9 MJ ME. At the end of the feeding period the ewes were slaughtered and blood samples and ovaries were collected from individual animals. The numbers of large- (≤ 4 mm diameter) and small- to medium-sized (1 to 3 mm diameter) follicles were recorded and follicular fluids from both size groups were stored for later analyses following centrifugation. Each mature corpus luteum (CL) was isolated from its ovary and sliced to provide a pair of ca. 0⋅3 mm diameter sections. These sections (mean = 163 (s.e.12) mg) were cultured separately (4 h; 39°C; 5% CO2) in 3 ml Medium 199 in the absence or presence of 100 μIU LH. Dietary supplementation of calcium soaps of fatty acids increased progesterone concentrations in the plasma (P < 0⋅05) and follicular fluid (P < 0⋅01 and P < 0⋅05 for large and small to medium follicles, respectively); trigylcerides in plasma (P < 0⋅05); total cholesterol in plasma (P < 0⋅001) and follicular fluid (P < 0⋅001); HDL-cholesterol in plasma (P < 0⋅001) and follicular fluid (P < 0⋅01) and LDL-cholesterol in plasma (P < 0⋅01). Progesterone released by luteal tissue into the culture medium in vitro from ewes given protected lipid was higher than that from tissue of control ewes (P < 0⋅05) and supplementary LH in vitro stimulated progesterone secretion by luteal tissues from both groups (P < 0⋅05). In conclusion, a dietary supplement of calcium soaps of fatty acids altered the intra-follicular environment surrounding the oocyte and follicular cells and enhanced luteal function in sheep.

Energy metabolism during flushing of mink (Mustela vison) in relation to a control group was studied by means of combining pooled data from balance and respiration experiments carried out in 1993 (experiment I) and 1994 (experiment II) and some blood metabolites from experiment II. The experiments, which were divided into six 1-week balance periods, started on 7 February and continued until 22 March. Flushing was performed by restricted feeding in periods 2 and 3 and refeeding in periods 4 and 5. Each balance period included a 24-h measurement of heat production by means of indirect calorimetry in open-air circuit respiration chambers. In experiment II blood samples were taken at weekly intervals and analysed for total triiodothyronine, total thyroxine, free thyroxine, glucose, insulin and fructosamine. Generally, intake of digestible nutrients and energy metabolism measurements within the flushed group were strongly influenced by period (energy supply), whereas they remained stable in the control group and differences between treatment groups over the total experiment were non-significant. The fluctuation in energy status induced by flushing did not involve major changes in body weight. Blood metabolites reflected the food intake by being fairly stable in the control group and decreasing during restriction and increasing during refeeding in the flushed group (P < 0·05). As an effect of flushing the number of corpora lutea increased significantly (P < 0·05).

Ovaries from adult cycling rats were studied from 1600 h on the day of prooestrus to 0700 h on the day of oestrus in order to relate the cyclic hormonal changes to the proliferative activity of preovulatory and postovulatory (i.e. newly-formed corpora lutea) follicles. Proliferative activity was studied by the immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine (BrdU). The proliferative activity of granulosa cells (GC) in large preovulatory follicles showed a centripetal pattern and decreased during prooestrus, reaching a minimum at 2100 h. However, a proliferative wave was found in the GC of preovulatory follicles at 0200 h on the day of oestrus and in those of newly-formed corpora lutea at 0700 h on the day of oestrus. These results suggest that the granulosa cells of preovulatory follicles show maturational changes that followed a different pattern, depending on their location within the follicle, and that the proliferative wave found from 0200 to 0700 h on oestrus is important for the establishment of the number of steroidogenic cells in the cyclic corpus luteum.