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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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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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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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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.

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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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P R S Santos, F D Oliveira, M A M Arroyo, M F Oliveira, P Castelucci, A J Conley, and A C Assis Neto

The androgen/estrogen balance is essential for normal sexual development and reproduction in mammals. Studies performed herein investigated the potential for estrogen synthesis in cells of the testes of a hystricomorph rodent, Galea spixii. The study characterized the expression of the key enzymes responsible for estrogen and androgen synthesis, cytochromes P450 aromatase (P450arom), 17α-hydroxylase/17,20-lyase (P450c17) respectively, as well as the redox partner NADPH cytochrome P450 oxido-reductase (CPR) required to support electron transfer and catalysis of these P450s, by immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) analysis, throughout postnatal sexual development. Testes (immature, pre-pubertal, pubertal and post-pubertal) were collected, fixed for IHC (CYP19, CYP17 and CPR) and stored frozen for qPCR for the relevant gene transcripts (Cyp19a1 and Cyp17a1). Expression of P450c17 was significantly elevated at the pre-pubertal and pubertal stages. Based on IHC, P450c17 was expressed only in Leydig cell clusters. The expression of P450arom was detectable at all stages of sexual development of Galea spixii. IHC data suggest that estrogen synthesis was not restricted to somatic cells (Leydig cells/Sertoli cells), but that germ cells may also be capable of converting androgens into estrogens, important for testicular function and spermatogenesis.

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P Browne, N J Place, J D Vidal, I T Moore, G R Cunha, S E Glickman, and A J Conley

Female spotted hyenas (Crocuta crocuta) have an erectile peniform clitoris and a pseudoscrotum but no external vagina, all established by day 35 of a 110-day gestation. Recent studies indicate that these events are androgen-independent, although androgen secretion by fetal ovaries and testis was hypothesized previously to induce phallic development in both sexes. We present the first data relating to the capacity of the ovaries and testes of the spotted hyena to synthesize androgens at different stages of fetal life. Specifically, spotted hyena fetal gonads were examined by immunohistochemistry at GD 30, 45, 48, 65, and 95 for androgen-synthesizing enzymes, as related to the morphological development. Enzymes included 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5, 3β-hydroxysteroid dehydrogenase (3βHSD), and cholesterol side-chain cleavage cytochrome P450 (P450scc). Anti-Müllerian-hormone (AMH) expression was also examined. AMH was strongly expressed in fetal Sertoli cells from GD 30 and after. P450c17 expression was detected in Leydig cells of developing testes and surprisingly in Müllerian duct epithelium. Fetal ovaries began to organize and differentiate by GD 45, and medullary cells expressed P450c17, cytochrome b5, 3βHSD, and P450scc. The findings support the hypothesis that external genital morphology is probably androgen-independent initially, but that fetal testicular androgens modify the secondary, male-specific phallic form and accessory organs. Fetal ovaries appear to develop substantial androgen-synthesizing capacity but not until phallic differentiation is complete, i.e. after GD 45 based on circulating androstenedione concentrations. During late gestation, fetal ovaries and testes synthesize androgens, possibly organizing the neural substrates of aggressive behaviors observed at birth in spotted hyenas. These data provide an endocrine rationale for sexual dimorphisms in phallic structure and reveal a potential source of androgenic support for neonatal aggression in female and male C. crocuta.

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S C Loux, A J Conley, K E Scoggin, H El-Sheikh Ali, P Dini, and B A Ball

Steroid production varies widely among species, with these differences becoming more pronounced during pregnancy. As a result, each species has its own distinct pattern of steroids, steroidogenic enzymes, receptors, and transporters to support its individual physiological requirements. Although the circulating steroid profile is well characterized during equine pregnancy, there is much yet to be explored regarding the factors that support steroidogenesis and steroid signaling. To obtain a holistic view of steroid-related transcripts, we sequenced chorioallantois (45 days, 4 months, 6 months, 10 months, 11 months, and post-partum) and endometrium (4 months, 6 months, 10 months, 11 months, and diestrus) throughout gestation, then looked in-depth at transcripts related to steroid synthesis, conjugation, transportation, and signaling. Key findings include: 1) differential expression of HSD17B isoforms among tissues (HSD17B1 high in the chorioallantois, while HSD17B2 is the dominant form in the endometrium) 2) a novel isoform with homology to SULT1A1 is the predominant sulfotransferase transcript in the chorioallantois; and 3) nuclear estrogen (ESR1, ESR2) and progesterone (PGR) expression is minimal to nonexistant in the chorioallantois and pregnant endometrium. Additionally, several hypotheses have been formed, including the possibility that the 45-day chorioallantois is able to synthesize steroids de novo from acetate and that horses utilize glucuronidation to clear estrogens from the endometrium during estrous, but not during pregnancy. In summary, these findings represent an in-depth look at equine steroid-related transcripts through gestation, providing novel hypotheses and future directions for equine endocrine research.