G. J. KING
HELEN McMENAMIN and G. J. KING
Following treatment with gonadotrophins (Casida, 1935), pregnancy can be initiated in prepubertal gilts but the corpora lutea regress and gestation is not maintained for more than 20 to 25 days (Dziuk & Gehlbach, 1966). It has been suggested (Shaw, McDonald & Baker, 1970) that the uterus in the immature animal may be insufficiently developed to allow proper implantation and to prevent uterine luteolysis. Ovarian steroids are required throughout the prepubertal period for normal uterine growth (R. G. Brown and G. J. King, unpublished observations) but uterine weight increases more rapidly after the first oestrus (Dyck, 1971). In an attempt to develop the prepubertal uterus and thus increase the chance of maintaining pregnancy, eight gilts (treated group) were injected with 1000 i.u. PMSG followed 72 hr later by 800 i.u. HCG at approximately 100, 121
G. Cassar, W. A. King and G. J. King
Nineteen gilts were used in an experiment to examine the relationship between rate of development and embryonic sex on day 10 of pregnancy. All gilts were mated to the same boar approximately 24 h after detection of second oestrus. They were individually housed and fed similar diets until slaughter on day 10 of gestation (day 0 = day of insemination) for subsequent recovery of the conceptus. All conceptuses were photographed and their surface areas (mm2) measured by tracing outlines on a digitized tablet interfaced with a computer program. Within each litter, individuals were categorized as small, medium or large by three equal divisions of the size range between the smallest and largest member. Conceptuses were individually cultured in Medium 199 with 1% colcemid and stained with 4% Giemsa. Metaphase spreads were located and sex was determined by presence or absence of the Y chromosome in at least two spreads from each specimen. A total of 214 conceptuses were recovered but only 125 (58%) were successfully karyotyped. The overall sex ratio was not significantly different from 1:1 (57 males and 68 females; P> 0.25). Sex was determined in 51 of 88 small embryos, 22 of 44 medium embryos and 52 of 82 large embryos and males represented 9 (17.6%), 10 (45.5%) and 38 (73%), respectively. Logistic analysis indicated significantly more females in the small and significantly more males in the large groups (P < 0.001). Results demonstrate that most male conceptuses grow faster than females before commencement of attachment to the uterine lining.
G. K. Nessan and G. J. King
Summary. Sexual behaviour was not induced in ovariectomized cows by single doses of 100 or 250 pg oestradiol benzoate or by 3 injections of 50 μg oestradiol benzoate given at 12-h intervals. Treatment with 3 doses of 250 or 500 μg oestradiol benzoate at 12-h intervals produced plasma levels that were similar to those during pro-oestrus and oestrus in intact cows, and 11 of 12 treated animals showed oestrous behaviour. The higher dose resulted in a more pronounced response in 'mounting' and 'mounted' behaviour and a shorter interval to the onset of behavioural signs of oestrus. Oestrous behaviour was not observed in any ovariectomized cows treated with 1, 2 or 20 mg testosterone propionate, doses that resulted in plasma levels comparable to those in intact pro-oestrous animals. Treatment with pharmacological doses of testosterone propionate resulted in transient mounting and interest in similarly treated females, but the results were inconsistent.
J. P. Laforest and G. J. King
Summary. The permeability of subepithelial capillaries in porcine endometrium was studied during midcycle and early pregnancy. Gilts were slaughtered on Day 13 or Day 15 of the oestrous cycle or pregnancy, 15 min after injection through the ear vein of 11·1 MBq of 125I-labelled human albumin in phosphate-buffered saline. The radio-activity of endometrial strips taken along the mesometrial and antimesometrial aspects of the uterine horn varied on average from 270 to 701 c.p.m./g and no difference (P > 0·05) was found between reproductive status, days of slaughter or sampling sites. The majority of the subepithelial capillaries showed ultrastructural evidence of increased vascular permeability, such as marked thinning of the capillary walls, especially on the side proximal to the epithelial basal lamina, multilayering and partial disparition of the endothelial basal lamina and abundant endothelial vesicles. Fenestrated pores were observed, but were rare. There was no obvious difference between reproductive status, days of sampling or sampling sites inside the uterus, suggesting that on Days 13–15 after oestrus the ultrastructural characteristics of porcine endometrial capillaries are little affected by the presence of attaching blastocysts and supporting the results obtained with radioactive albumin. Ferritin injected directly into a uterine artery of one gilt on Day 15 of pregnancy was carried through the capillary wall by endothelial vesicles, showing ultrastructural evidence of increased permeability.
Keywords: ferritin; radioactive albumin; endometrium; vascular permeability; pig
J. L. Keys and G. J. King
The ultrastructure of subepithelial capillaries in pig endometrium was studied after intrauterine and systemic oestradiol treatment. Gilts were killed on day 13 or 19 (n = 4 per day per treatment) after surgical introduction of Silastic beads containing either oestradiol or cholesterol into the uterine lumina on day 10 after oestrus. An additional group of gilts was injected i.m. with 5 mg oestradiol valerate on days 11–15 of the oestrous cycle and killed on day 13 or day 19 (n = 4 per day). Light and electron microscope studies of endometrial samples revealed that cholesterol beads did not appear to affect subepithelial capillary structure. Both intrauterine and systemic treatment of gilts with oestradiol were associated with regional modification of the capillary wall involving attenuation and fenestration of the face directly underlying the uterine epithelium, accumulation of caveolae in the opposite face of the capillary, and the occurrence of discontinuous, multilayered capillary basal laminae. The similarity of these structural changes to those observed in capillaries at the time of embryonic attachment suggests that oestrogens of blastocyst origin may function to modify capillary morphology and possibly facilitate increased transcapillary traffic during the establishment of pregnancy.
J. P. Laforest and G. J. King
Summary. Silastic beads were inserted into the uterine lumen on Day 10 after oestrus. Gilts received beads containing oestradiol-17β only, oestradiol benzoate, or oestradiol-17β + prostaglandin (PG) E-2. Oestrous cycles were slightly longer in treated than in untreated pigs (20·2 ± 0·4 days), and durations were 22·6 ± 1·3, 26·2 ± 1·7 and 23·2 ± 1·8 days for oestradiol-17β, oestradiol benzoate and oestradiol-17β + PGE-2 treatments, respectively (P > 0·05). Thus, PGE-2 and an oestrogen such as oestradiol benzoate that persist for a longer period cannot prolong the cycle more than oestradiol-17β alone. Additional cyclic gilts underwent similar treatments with beads containing oestradiol-17β, oestradiol-17β + PGE-2 or cholesterol, and cannulation of one utero-ovarian vein on Day 10. Blood samples were collected from the catheter every 15 min from 08:00 until 11:00 h and from 20:00 until 23:00 h for 5 consecutive days starting the day after surgery and peripheral plasma samples were also collected daily. On Day 16, beads containing oestradiol-17β were surrounded by endometrial folds whereas cholesterol beads were free. Concentrations of plasma progesterone did not vary significantly from Days 11 to 16 in gilts treated with oestradiol-17β or oestradiol-17β + PGE-2, but decreased in cholesterol-treated gilts. Concentrations of plasma oestrone and oestradiol-17β were more than ten times higher in gilts treated with oestradiol-17β or oestradiol-17β + PGE-2 than in cholesterol-treated gilts on the day after bead insertion, but decreased rapidly to values comparable to those in cholesterol-treated gilts by Day 14. In contrast, concentrations of oestrone sulphate remained high until Day 16. Concentrations of PGE-2 in the utero–ovarian vein plasma did not differ (P > 0·05) between treatments but those of PGF-2α were higher (P < 0·004) in gilts treated with cholesterol than in those treated with oestradiol-17β or oestradiol-17β + PGE-2. It is postulated that insufficient oestradiol-17β is released by the beads toward the end of a 'recognition period' to prolong the cycle for more than 3–6 days
Keywords: oestradiol-17β; prostaglandin E-2; luteolysis; oestrous cycle; pig
G. J. King and C. A. Ackerley
Summary. Segments of individual blastocysts collected on Days 10, 12, 14 and 16 were examined microscopically to observe yolk-sac development and treated immunocytochemically to localize oestrogens in specific membranes. Mesoderm was present beneath the embryonic disc of ovoid blastocysts on Day 12. The mesoderm spread beyond 1 cm from the disc on Day 14, producing a splanchnic yolk-sac membrane extending across the blastocoelomic cavity, but no mesodermal cells had yet reached 5 cm. By Day 16, proliferation of mesoderm and development of the yolk sac had progressed beyond 20 cm from the disc in most of the specimens examined. Incubation of ultrathin sections with sheep antiserum to oestrone or oestradiol-17β followed by rabbit anti-ovine IgG—gold complex and subsequent counting of gold particles retained over the tissues gave a weakly positive reaction for oestrone in trophectodermal cells on Day 10. The most intense reaction for oestradiol-17β was also present in the trophectoderm and yolk-sac endoderm on Days 12, 14 and 16.
L. A. Bate and G. J. King
Summary. Pig blastocysts aged 14, 16 and 18 days were divided into 15 cm segments representing tissue adjacent to the embryonic disc, an intermediate section and the tip region. Whenever total blastocyst length allowed, the intermediate segment was divided into proximal and distal portions for separate culture. All were rinsed with buffer and incubated with dehydroepiandrosterone for 3 h. Rinsing buffer and incubation medium were subsequently assayed for concentrations of oestrone and oestradiol-17β. The highest production of oestrogen was found in the embryonic disc region. The intermediate regions had the lowest synthetic ability, while the tip region produced more oestrogens than the intermediate regions but less than the disc region. The production of oestrone was higher (P < 0·05) in 18-day-old blastocysts than in younger ones while oestradiol-17β production was lower (P < 0·05) on Day 16. The proportional role of the embryonic disc region as oestrogen-producing tissue increased over time. On Day 14, each intermediate region produced over 70% as much oestrogen as the disc region. These proportions declined on Days 16 and 18 to about 50 and 30% respectively. The regional variation in the ability of blastocysts to produce oestrogens may have some influence on the ability of the blastocyst to create an adequate microenvironment within the uterus which permits successful differentiation and placentation.
Keywords: oestrogens; blastocyst; pigs; pregnancy; uterine environment
H. A. ROBERTSON and G. J. KING
The changes in the plasma levels of progesterone, of unconjugated oestrone and oestradiol-17 β, and of oestrone sulphate in the sow at the time of implantation, during gestation and at parturition are described. The progesterone concentration remained fairly constant (10 to 12 ng/ml) throughout pregnancy until 20 to 15 days before parturition. The progesterone concentration dropped over the last 15 days of gestation. Measurable quantities of oestrone sulphate were present in the maternal plasma at Day 16 but not at Day 9 of gestation and rose to a peak >3 ng/ml (assessed as free oestrone) between Day 23 and Day 30. The concentration fell to a low level of 35 pg/ml on Day 46 before rising to a second peak of 3 ng/ml just before parturition. Measurable quantities of unconjugated oestrone (>10 pg/ml) and oestradiol-17 β (>15 pg/ml) first appeared in the plasma at approximately Day 70 to Day 80 and their rise to a peak at the time of parturition followed very closely that of oestrone sulphate. Unconjugated oestrone rose to a peak at a mean concentration of 2·5 ng/ml and oestradiol-17 β at 400 pg/ml. No oestradiol-17α was detected. The concentration of these steroids returned to basal levels by 72 hr after parturition.