Heather L. Christie

Estrogen Metabolism in the Equine Conceptus and Endometrium During Early Pregnancy in Relation to Estrogen Concentrations in Yolk-Sac Fluid

Abstract Because estradiol (E2) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E2, estrone (E1), and their sulfates (E2S, E1S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11–26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E1S. Then, the metabolism of E2, which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus. Eleven conceptuses collected mainly on Days 12, 15, and 18, with endometrial biopsy samples taken immediately thereafter, were used for steroid metabolic studies. Trophoblastic and endometrial tissues were incubated with [3H]-labeled E2 or E1, and with [14C]-E1 in one experiment. Steroids were recovered from the media by solid-phase extraction (SPE) and eluted separately as unconjugated and conjugated fractions. Conjugation increased from Day 12 for the trophoblast (more so by bilaminar than trilaminar tissues on Day 18) and was much greater for endometrium, with almost all as sulfoconjugates. HPLC profiles of free and sulfate fractions were obtained from a gradient of acetonitrile/water. Interconversion (E2 ⇌ E1) by trophoblast varied with development; it favored E2 in older conceptuses, more in bilaminar than trilaminar tissues. Some more polar products were also noted, with loss of tritium seen as [3H]2O at SPE, and confirmed by HPLC in a second system with authentic reference steroids. Almost all radioactivity in the endometrium was present as E2 in both free and sulfate fractions. It was concluded that local metabolism of E2 is quantitatively significant and may play an important role in the actions of the large amounts of estradiol produced by the early equine conceptus.

The in vitro metabolism of cortisol by ovarian follicles of rainbow trout (Oncorhynchus mykiss): comparison with ovulated oocytes and pre-hatch embryos

Mid-vitellogenic stage rainbow trout (Oncorhynchus mykiss) ovarian follicles (both intact and yolk free (YF)), ovulated oocytes and embryos were co-incubated with [2,4,6,7-(3)H]cortisol for 18 h to determine the degree and nature of the metabolism and biotransformation of the glucocorticoid. There was evidence of the conversion of cortisol to the less biologically potent glucocorticoid, cortisone, and the formation of glucocorticoid sulphates (both cortisol and cortisone) for all cell and tissue samples, suggesting the presence of 11β-hydroxysteroid dehydrogenase (11β-HSD) and glucocorticoid sulphotransferase (GST) activity at all stages; however, GST activity was particularly marked in both intact and YF ovarian follicles, suggesting an important role of follicles in limiting the exposure of oocyte to maternal cortisol. As there was no evidence of 11β-HSD or GST activity in ovarian fluid, the findings affirm that ovarian follicles (probably the thecal and granulosa cells) provide a barrier against the transfer of cortisol to the oocytes by forming sulphated steroids, whereas ovulated oocytes and early embryos have a more limited capacity to either metabolize or conjugate cortisol and are therefore more vulnerable at the post-ovulatory and early embryonic stages to increases in exposure to the glucocorticoid.

Suitability of epididymal and testicular ultrasonography and computerized image analysis for assessment of current and future semen quality in the ram.

Breeding soundness evaluation (BSE) is the primary assessment for determining the reproductive potential of male animals. This method, however, cannot be used to evaluate semen frequently or to predict future semen quality. Computerized analysis of ultrasonographic images provides information on histophysiological changes in male reproductive organs. We hypothesized that: (i) semen parameters would correlate with ultrasonographic characteristics of the distal region (cauda) of the epididymis and (ii) testicular ultrasound images and/or circulating testosterone concentration would predict future semen quality in the ram. Six adult rams underwent BSE and scrotal ultrasonography approximately 60 d apart (average duration of the spermatogenic cycle) both during the breeding (December and February) and non-breeding (June and August) seasons. An inverse correlation was found between pixel intensity (numerical pixel values) of the epididymes and percentage of sperm in semen with normal morphology (r = −0.46, P < 0.05). Pixel heterogeneity (standard deviation of pixel values) correlated negatively with percentage of sperm with normal morphology (r = −0.42, P < 0.05) and directly with percentage of spermatozoa with abnormal tails (r = 0.43, P < 0.05). Pixel heterogeneity of testicular parenchyma obtained approximately 60 d prior to semen evaluation inversely correlated with percentage of sperm with normal morphology (r = −0.73, P < 0.01) and sperm progressive motility (r = −0.76, P < 0.01), and directly with percentage of sperm with abnormal tails (r = 0.72, P < 0.01) and loose heads (r = 0.79, P < 0.01). We concluded that scrotal ultrasonography combined with computer-assisted analyses of epididymal and testicular echotexture in the ram was a valuable method for determining certain current and future semen parameters, respectively.

Relationship between the timing of prostaglandin-induced luteolysis and effects on the conceptus during early pregnancy in mares

To advance the understanding of early pregnancy and pregnancy failure in horses, this study determined how luteolysis induced by cloprostenol (an analogue of prostaglandin F2α) affects conceptus development. Mares were injected on Days 12, 14, 16 or 18 of pregnancy with either cloprostenol (treatment groups, total n=83 pregnancies) or saline (controls, n=81), and growth of the conceptuses was monitored and compared by daily ultrasonography until they were collected transcervically on Days 15-22, 1-4 days after the injections. The comparisons were extended in the recovered conceptuses by counting somites, measuring the volume and osmolality of yolk-sac fluid and its concentrations of proteins, estrone sulfate and progesterone, and by assessing the morphology of the capsule and vascular system. When luteolysis was initiated on or before Day 16, most pregnancies survived until the time of collection and the conceptuses in respective treated and control groups on Days 15-20 were very similar except for some effects of treatment on the capsule and vascular development. In contrast, after luteolysis was initiated on Day 18, abortion often ensued within 3 days and most conceptuses collected had degenerated, therein constituting a predictable system in which to study the pathogenesis of a particular cause of pregnancy failure.

Estrogen metabolism by the equine embryo proper during the fourth week of pregnancy

Estrogen production by the trophoblast is considered important in early equine pregnancy and leads to high concentrations in yolk-sac (Y-S) fluid. The embryo proper is a potential site for their action. We examined estrogen metabolism in the embryo proper because some actions of estrogens are derived from locally formed metabolites. The embryo proper, as well as separated extraembryonic tissues, of conceptuses collected about day 25 of pregnancy, were incubated with (3)[H]-estrone (E(1)) and (3)[H]-estradiol (E(2)). Steroids were recovered from media by solid-phase extraction and eluted separately as unconjugated and conjugated fractions. Profiles of free and sulfo-conjugated fractions were obtained by HPLC. Some differences and similarities were noted for the embryo proper as compared to the extraembryonic tissues. No reduction of E(1) to E(2) was noted for the embryo proper and allantois, but some was seen with the bilaminar Y-S wall. Less conversion of E(2) to E(1) occurred in the embryo proper than in the extraembryonic tissues. Profiles for hydrolyzed sulfates from incubation of the embryo proper were very similar for both substrates, mainly with E(1) present. Thus, low levels of reductase and high levels of oxido- activities were apparent for the 17beta-hydroxysteroid dehydrogenase enzymes. Further evidence of an active role for the embryo proper was seen as minor, polar products, and an unknown compound eluting between E(2) and E(1). These findings show, for the first time, that the embryo proper can metabolize estrogens that are found in Y-S fluid - a function of potential significance at this stage in its development.

5Alpha-Reduced Steroids Are Major Metabolites in the Early Equine Embryo Proper and Its Membranes

ABSTRACT Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro- and 3beta,5alpha-tetrahydro- steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta,5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta,5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids—for example, 5alpha-dihydrotestosterone in male sexual differentiation—provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in early embryonic development in horses and, more generally, in other species.

The presence of 19-norandrostenedione and its sulphate form in yolk-sac fluid of the early equine conceptus

C(18) neutral steroid formation by cytochrome P450 aromatase has been recorded for several equine and porcine tissues. High activity of P450 aromatase is reflected in the quantities of estrogens in yolk-sac (y-s) fluid of early equine conceptuses. In a previous study of y-s fluid we detected large amounts of androgens by radioimmunoassay (RIA), using an antiserum for androstenedione (A(4)). Here, we report that RIA, following chromatography, gave tentative identification of the major peak as norandrostenedione (19-norA) not as A(4). Furthermore, even greater quantities of 19-norA seemed to be present in y-s fluid as a sulphoconjugate, as noted from extraction, solvolysis, HPLC, followed by RIA. Confirmation of these unusual findings was attained after further purification with two HPLC systems and definitive identification by LC-MS with an authentic standard of 19-norA. Initial extraction of the steroid sulphate as a methylene-blue complex also yielded 19-norA suggesting that the 3-enol form had enabled sulphoconjugation. The biological significance of retention mainly as a sulphate is not known; however, the large amounts of 19-norA found in the fluid accords well with reports on the catalytic activity shown in vitro by the blastocyst isozyme of P450 aromatase in the pig and horse.

Biosynthesis of oestrogen by the early equine embryo proper

The embryo proper in early equine pregnancy has recently been shown to have a remarkable capacity for metabolism of oestrogens. High concentrations of oestrogens in yolk-sac fluid could provide substrate for local metabolism in tissues of the embryo proper and this activity could have significance for early development. Due to the high level of oestrogen metabolism in the embryo proper we examined the possibility that it could also biosynthesise oestrogens. Conceptuses were collected in the fourth week of pregnancy (n=23) and the embryo was separated from extraembryonic tissues for incubation with [(3)H]androstenedione. Steroids were recovered from media by solid-phase extraction and eluted as unconjugated and conjugated fractions. Profiles of free and sulfoconjugated fractions, as well as the phenolic steroids extracted from them, were obtained by chromatography. Oestrone and oestradiol were seen clearly, indicating oestrogen biosynthesis, and the presence of more polar products, arising from metabolism of the primary oestrogens, gave further evidence that the embryo was capable of oestrogen biosynthesis. Aromatase activity was also demonstrated by detection of tritium loss, as (3)H(2)O, from incubations (n=3) with [1β-(3)H]androstenedione. It is suggested that its oestrogen biosynthesis may have significance for the remarkable development of the vasculature in the embryo proper at this stage.

Modulation of GR activity does not affect the in vitro metabolism of cortisol by rainbow trout ovarian follicles

The goal of the study was to determine whether the metabolic clearance of cortisol from rainbow trout (Oncorhynchus mykiss) ovarian follicles is affected by the level of ovarian steroidogenesis, and whether it involves the activation of glucocorticoid receptors (GRs). Ovarian follicles were incubated in vitro; the adenylate cyclase activator, forskolin, was used to stimulate ovarian steroidogenesis, and the modulation of GR activity was brought about using GR agonists (cortisol and dexamethasone) or the GR antagonist, mifepristone (RU486). The follicles were co-incubated with [2, 4, 6, 7 3H] cortisol, and the tritium-labelled steroid products were separated by HPLC. In addition, the rates of expression of genes encoding for the two forms of GR (gr1 and gr2) were measured. Cortisone, cortisol sulphate, and cortisone sulphate were the major glucocorticoid products of cortisol metabolism, indicative of the action of 11β-hydroxysteroid dehydrogenase and glucocorticoid sulphotransferase in the follicular cells. There were no effects of RU486 or forskolin on the rates of [3H]cortisol metabolism suggesting that cortisol metabolism by ovarian follicles was independent of GR activation, and not influenced by increased activation of gonadal reproductive steroidogenesis.

Patterns of conceptus development and of progesterone concentrations in maternal blood preceding spontaneous early pregnancy failure in mares

Sixteen cases of spontaneous pregnancy loss (11 of singletons and five of pairs of twins) are described. The losses occurred between gestation Days 13 and 25 in 12 mares being monitored almost daily by transrectal ultrasonography (for measurement of conceptus growth) and blood sampling (for determination of maternal plasma progesterone concentrations as evidence of luteolysis) in experimental studies of early pregnancy. In 10 of the 16 cases the uterus was flushed and eight conceptuses were recovered for morphological assessment. Five of the 11 losses of singletons occurred before Day 16 and, with one exception, were preceded or accompanied by luteolysis. The remaining six singleton pregnancies failed after Day 16, with two cases evidencing luteolysis beforehand. Thus, overall, 6/11 singleton losses were associated with luteolysis while 5/11 were not. The five cases of simultaneous loss or degeneration of twin conceptuses all occurred on Day 19 or 20, preceded by luteolysis in only one case. These observations suggest that while the causes of spontaneous early pregnancy failure are multifactorial, luteolysis might contribute to the problem more often than has been previously contended.