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J Almeida, A J Conley, L Mathewson and B A Ball

In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and estrogens. 3β-hydroxysteroid dehydrogenase/Δ54-isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. Androgens produced by P450c17 serve as substrate for estrogen synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3βHSD, and P450arom and to determine changes in expression during development in the prepubertal, postpubertal, and adult equine testis based upon immunohistochemistry (IHC) and real-time quantitative PCR. Based on IHC, 3βHSD immunolabeling was observed within seminiferous tubules of prepubertal testes and decreased after puberty. On the other hand, immunolabeling of 3βHSD was very weak or absent in immature Leydig cells of prepubertal testes and increased after puberty. HSD3B1 (3 β HSD gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal testes (P=0.0041). P450c17 immunolabeling was observed in small clusters of immature Leydig cells in prepubertal testes and increased after puberty. CYP17 (P450c17 gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.030) and postpubertal testes (P=0.0318). A weak P450arom immunolabel was observed in immature Leydig cells of prepubertal testes and increased after puberty. Similarly, CYP19 (P450arom gene) mRNA expression was higher in adult testes compared with prepubertal (P=0.0001) and postpubertal (P=0.0001) testes. In conclusion, Leydig cells are the primary cell type responsible for androgen and estrogen production in the equine testis.

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M. A. Kaminski, S. P. Ford and A. J. Conley

The expression of cytochromes P450 17α-hydroxylase (P450c17) and aromatase (P450arom) was compared between preimplantation Chinese Meishan and domestic Yorkshire conceptuses during the period encompassing maternal recognition of pregnancy. Individual conceptuses were recovered on days 10.5, 11.0, 11.5, 12.0, and 14.0 of gestation. Diameter (spherical blastocysts), length (elongated blastocysts), DNA, protein and oestradiol content, as well as the amounts of P450c17 and P450arom (western analysis) were determined in individual conceptuses. Comparisons were made only between conceptuses of similar diameters on each day which restricted analyses to blastocysts 6 mm or less in diameter on days 10.5–12.0. Nonetheless, both DNA and protein content were greater in Yorkshire than in Meishan conceptuses. Oestradiol content also tended to be greater in Yorkshire than in Meishan conceptuses across days. A significant effect of breed and breed by day interaction was detected for P450c17. Expression of P450c17 in Yorkshire conceptuses increased markedly above that in Meishan conceptuses by day 11, remained high until day 11.5 and returned to values similar to those of Meishans by day 12. The expression of P450arom was also greater in Yorkshire than in Meishan conceptuses, but no breed by day interaction was detected. These data suggest that differences in development between Meishan and Yorkshire conceptuses include trophoblastic differentiation during preattachment stages. The significance and impact of this divergence in development on subsequent growth and survival remains to be determined.

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Alan J Conley and Barry A Ball

Historically, studies on the endocrinology of pregnancy and parturition in horses have made major contributions of relevance to mammals in general. Recent use of liquid chromatography mass spectrometry, measuring multiple steroid hormones simultaneously in blood, foetal and placental tissues throughout normal gestation, and in mares with experimentally induced placentitis, has advanced our current understanding of many of the unusual strategies seen during gestation and at foaling. This includes the stimulation of luteal steroidogeneisis by equine chorionic gonadotropin (eCG) from the endometrial cups, resulting in additional androgen and oestrogen secretion. Progesterone declines as the endometrial cups and eCG disappears, replaced by 5α-dihydroprogesterone (DHP), a potent equine progesterone receptor (PR) agonist, as the chorioallantoic placenta develops. Placental steroidogenesis thereafter is influenced by foetal pregnenolone and dehydroepiandrosterone secretion, providing substrate for 5α-pregnane and oestrogen synthesis, an unusual example of a ‘foeto-placental unit’. Foetal gonadal dehydroepiandrosterone fuels placental oestrone sulphate secretion, peaking at higher concentrations in mares than any other species known, declining steadily thereafter to term. Additional 5α-reduced (DHP) metabolites increase from mid-gestation to peak concentrations 3–5 days before foaling, declining prepartum, most likely as a result of selective loss of placental SRD5A1 (5α-reductase) expression and activity. Similar changes occur in mares with experimentally induced placentitis, which is also associated with a decreased ratio of equine PR-B:PR-A in myometrium, suggesting that progestin withdrawal is both systemic (pregnanes) and local (receptor-dependent) in mares. In addition, some steroids detected during equine pregnancy by immuno-assay are not detected by mass spectrometry, further illustrating the immense value of this technology.

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M. A. Kaminski, S. P. Ford, C. R. Youngs and A. J. Conley

The effect of sex on pig conceptus development to day 12 of gestation was investigated. On day 2 of gestation, reciprocal embryo transfers were performed resulting in four groups (Yorkshire–Yorkshire, Yorkshire–Meishan, Meishan–Yorkshire and Meishan–Meishan). Conceptuses at day 12 were recovered from each recipient and diameter, as well as DNA, protein and oestradiol content were determined for individual conceptuses. The sex of individual conceptuses at day 12 was determined by amplification of a fragment of the pig SRY gene, using the polymerase chain reaction. Embryos developed more rapidly to day 12 in Yorkshire recipients, but there was no detectable effect of sex on the diameter, DNA, protein or oestradiol content of conceptuses from any transfer group. Thus, no sex effect was apparent under conditions either promoting or retarding the rate of early pig blastocyst growth. These results provide strong evidence that pig embryonic development occurs at a rate determined by uterine environment and not by sex of the conceptus.

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S. P. Ford, N. K. Schwartz, M. F. Rothschild, A. J. Conley and C. M. Warner

Summary. Embryonic cell number in miniature pigs inbred for specific SLA haplotypes (a, c, and d) was determined on Day 6 by nuclear staining and, on Days 9 and 11, by DNA analyses (first day of oestrus = Day 0). Pigs exhibiting first behavioural oestrus at 08:00 h were hand-mated to an SLA homozygous boar 12 and 24 h later. Numbers of embryos flushed from uteri at 08:00–10:00 h on Days 6, 9 and 11 were greater (P < 0·05) for SLAd females than for SLAa or SLAc females, which did not differ (8·2 vs 6·8 and 6·2, respectively). Recovery rates (embryos recovered/CL number) were similar, averaging 75·8% for all three SLA haplotypes. Embryos from SLAd dams contained fewer blastomeres (23 cells) on Day 6 than did embryos from SLAa (89 cells) or SLAc (79 cells) females. The reduced cell numbers of SLAd vs SLAa or SLAc embryos continued to Day 9 (28 vs 107 and 67 ng DNA/embryo) and Day 11 (167 vs 674 and 586 ng DNA/embryo). These results suggest an effect of the SLA complex on preimplantation embryonic development.

Keywords: pig; SLA complex; preimplantation; embryonic development

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Erin L Legacki, C J Corbin, B A Ball, M Wynn, S Loux, S D Stanley and A J Conley

Mammalian pregnancies need progestogenic support and birth requires progestin withdrawal. The absence of progesterone in pregnant mares, and the progestogenic bioactivity of 5α-dihydroprogesterone (DHP), led us to reexamine progestin withdrawal at foaling. Systemic pregnane concentrations (DHP, allopregnanolone, pregnenolone, 5α-pregnane-3β, 20α-diol (3β,20αDHP), 20α-hydroxy-5α-dihydroprogesterone (20αDHP)) and progesterone) were monitored in mares for 10days before foaling (n=7) by liquid chromatography–mass spectrometry. The biopotency of dominant metabolites was assessed using luciferase reporter assays. Stable transfected Chinese hamster ovarian cells expressing the equine progesterone receptor (ePGR) were transfected with an MMTV-luciferase expression plasmid responsive to steroid agonists. Cells were incubated with increasing concentrations (0–100nM) of progesterone, 20αDHP and 3α,20βDHP. The concentrations of circulating pregnanes in periparturient mares were (highest to lowest) 3α,20βDHP and 20αDHP (800–400ng/mL respectively), DHP and allopregnanolone (90 and 30ng/mL respectively), and pregnenolone and progesterone (4–2ng/mL). Concentrations of all measured pregnanes declined on average by 50% from prepartum peaks to the day before foaling. Maximum activation of the ePGR by progesterone occurred at 30nM; 20αDHP and 3α,20βDHP were significantly less biopotent. At prepartum concentrations, both 20αDHP and 3α,20βDHP exhibited significant ePGR activation. Progestogenic support of pregnancy declines from 3 to 5days before foaling. Prepartum peak concentrations indicate that DHP is the major progestin, but other pregnanes like 20αDHP are present in sufficient concentrations to play a physiological role in the absence of DHP. The authors conclude that progestin withdrawal associated with parturition in mares involves cessation of pregnane synthesis by the placenta.

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Erin L Legacki, Elizabeth L Scholtz, Barry A Ball, Scott D Stanley, Trish Berger and Alan J Conley

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (n=9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6–20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all Δ4 steroids. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (Δ5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus Δ4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5α-pregnanes) marked the luteo-placental shift, Δ5 steroids defined fetal gonadal growth and 5α-reduced metabolites of DHP dominated the steroid profile in mid- to late-gestation. Comprehensive LC–MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development.

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Erin L Legacki, C Jo Corbin, Barry A Ball, Kirsten E Scoggin, Scott D Stanley and Alan J Conley

Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) crucial for progesterone and androgen synthesis, respectively, as well as aromatase cytochrome P450 (P450arom) that converts Δ4-androgens to estrogens. 5α-reductase is another important enzyme in equine placentas because 5α-dihydroprogesterone (DHP) sustains pregnancy in the absence of progesterone in the second half of equine pregnancy. DHP and its metabolites decline dramatically days before foaling, but few studies have investigated placental enzyme activity before or at parturition in mares. Thus, key enzyme activities and transcript abundance were investigated in equine placentas at 300 days of gestation (GD300) and post-partum (term). Equine testis was used as a positive control for P450c17 activity. Substrates were incubated with microsomal preparations, together with enzyme inhibitors, and products were measured by liquid chromatography tandem mass spectrometry or radiometric methods (aromatase). Equine placenta expressed high levels of 3βHSD, 5α-reductase and aromatase, and minimal P450c17 activity at GD300 compared with testis (600-fold higher). At foaling, 3βHSD and aromatase activities and transcript abundance were unchanged but 5α-reductase (and P450c17) was no longer detectable (P < 0.05) and transcript was decreased. Trilostane inhibited 3βHSD significantly more in testis than placenta, suggesting possible existence of different 3βHSD isoforms. Equine placentas have significant capacity for steroid metabolism by 5α-reductase, 3βHSD and aromatase but little for androgen synthesis lacking P450c17. Declining pre-partum 5α-reduced pregnane concentrations coincide with selective loss of placental 5α-reductase activity and expression at parturition in horses.

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A. J. Conley, Y. C. Jung, N. K. Schwartz, C. M. Warner, M. F. Rothschild and S. P. Ford

Summary. Systemic blood was collected from and surgery performed on sows of 3 strains of miniature swine bred for specific SLA (swine MHC) haplotypes (a, c and d) from Day 2 to Day 6 after mating (first day of mating = Day 0). Ovulation rate was determined by counting corpora lutea and embryos were flushed from the uterus. Progesterone, oestradiol-17β and oestrone were quantitated in blood plasma and uterine flushings by RIA. SLAd/d females had a higher ovulation rate than SLAa/a or SLAc/c females (11·50 ± 0·87 vs 9·11 ± 0·68 and 8·17 ± 0·83, respectively; P < 0·01). Oestrone was higher than oestradiol-17β in systemic plasma (56·5 ± 6·4 vs 33·0 ± 4·7 pg/ml, P < 0·01) while oestradiol-17β was higher than oestrone in uterine flushings (19·8 ± 1·4 vs 14·9 ± 1·5 pg/horn, P < 0·10). Systemic progesterone concentration was correlated with day after mating (r = 0·93, P < 0·01). There was no effect of haplotype on any of the hormone concentrations measured. Litter size was analysed from 99 matings amongst SLAa/a, SLAa/c, SLAa/d, SLAd/c and SLAd/d sires and dams. Litter size from -/d and d/d sows or from d/d boars were larger (P < 0·05) than for all other matings. Although ovulation rate was higher in SLAd/d sows, the significant effect of sire SLA genotype on litter size suggests an additional effect of the d haplotype on embryonic survival.

Keywords: pig; major histocompatibility complex; litter size

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G Schuler, G R Özalp, B Hoffmann, N Harada, P Browne and A J Conley

No definitive information is yet available on the steroidogenic capacity of the two morphologically distinct cell types forming the bovine trophoblast, the uninucleated trophoblast cells (UTCs) and the trophoblast giant cells (TGCs). Hence, in order to localise 17α-hydroxylase-C17,20-lyase (P450c17) on a cellular level and to monitor its expression as a function of gestational age, placentomes from pregnant (days 80–284; n = 19), prepartal (days 273–282; 24–36 h prior to the onset of labour; n = 3) and parturient cows (n = 5) were immunostained for P450c17 using an antiserum against the recombinant bovine enzyme. At all stages investigated, P450c17 was exclusively found in the UTCs of chorionic villi (CV), where staining was ubiquitous between days 80 and 160, but was largely restricted to primary CV and the branching sites of secondary CV between days 160 and 240. Thereafter, a distinct ubiquitous staining reoccurred in the UTCs of all CV in late pregnant, prepartal and parturient animals. Using an antiserum against human aromatase cytochrome P450 (P450arom), specific cytoplasmic staining was observed in TGCs. In placentomes from pregnant cows, staining intensity was higher in mature compared with immature TGCs and was more pronounced in the trophoblast covering big stem villi compared with the trophoblast at other sites of the villous tree. In placentomes of a parturient cow, specific staining was only found in mature TGCs that survived the normal, but substantial, prepartal decline in TGC numbers. These results clearly showed that bovine UTCs and TGCs exhibit different steroidogenic capacities, constituting a ‘two-cell’ organisation for oestrogen synthesis. P450c17 expression appears to be quickly down-regulated and P450arom is up-regulated when UTCs enter the TGC differentiation pathway.