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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G Schuler, G R Özalp, B Hoffmann, N Harada, P Browne, and A J Conley
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.
C Passaro, D Tutt, D J Mathew, J M Sanchez, J A Browne, G B Boe-Hansen, T Fair, and P Lonergan
The objectives of this study were (i) to determine whether blastocyst-induced responses in endometrial explants were detectable after 6- or 24-h co-culture in vitro; (ii) to test if direct contact is required between embryos and the endometrial surface in order to stimulate endometrial gene expression; (iii) to establish the number of blastocysts required to elicit a detectable endometrial response; (iv) to investigate if upregulation of five interferon-stimulated genes (ISGs) in the endometrium was specific to the blastocyst stage and (v) to test if alterations in endometrial gene expression can be induced by blastocyst-conditioned medium. Exposure of endometrial explants to Day 8 blastocysts in vitro for 6 or 24 h induced the expression of ISGs (MX1, MX2, OAS1, ISG15, RSAD2); expression of IFNAR1, IFNAR2, NFKB1, IL1B, STAT1, LGALS3BP, LGALS9, HPGD, PTGES, ITGB1, AKR1C4, AMD1 and AQP4 was not affected. Culture of explants in the presence of more than five blastocysts was sufficient to induce the effect, with maximum expression of ISGs occurring in the presence of 20 blastocysts. This effect was exclusive to blastocyst stage embryos; oocytes, 2-cell embryos or Day 5 morulae did not alter the relative abundance of any of the transcripts examined. Direct contact between blastocysts and the endometrial surface was not required in order to alter the abundance of these transcripts and blastocyst-conditioned medium alone was sufficient to stimulate a response. Results support the notion that local embryo–maternal interaction may occur as early as Day 8 of pregnancy in cattle.
C Passaro, D Tutt, S Bagés-Arnal, C Maicas, R Laguna-Barraza, A Gutierrez-Adán, J A Browne, D Rath, S K Behura, T E Spencer, T Fair, and P Lonergan
The aims of this study were (i) to investigate changes in the global transcriptome of bovine endometrial explants induced by exposure to blastocysts, (ii) to investigate if male and female blastocysts elicit a differential response in the endometrial transcriptome in vitro and (iii) to determine whether bovine endometrium responds to the presence of murine embryos. In Experiment 1, endometrial explants from the same uterus were cultured for 6 h with or without 20 in vitro-produced bovine blastocysts. In Experiment 2, endometrial explants were cultured with male or female bovine blastocysts produced in vitro by IVF either using sex-sorted semen or conventional unsorted semen followed by embryo sexing based on a biopsy. In Experiment 3, endometrial explants were cultured alone or in the presence of bovine blastocysts (n = 25) or murine blastocysts (n = 25). Following culture, explants were snap frozen and stored at −80°C until RNA extraction, qPCR or RNA-Seq. Culture with bovine blastocysts increased endometrial expression of 40 transcripts, all of which were interferon-tau induced. Culture with male or female bovine blastocysts increased transcript abundance of five classic interferon-stimulated genes (MX1, MX2, ISG15, OASY1, RSAD2) in explants; however, there was no difference in abundance of transcripts previously reported to be related to embryonic sex (IFNAR1, IFNAR2, CTGF, ARTN, SLC2A1, SLC2A5). Exposure to murine blastocysts did not elicit any detectable change in transcript abundance. These findings, coupled with our previous data, indicate that very local, interferon-tau-induced changes in endometrial gene expression occur in response to blastocysts; whether such changes play any role in subsequent pregnancy recognition remains to be established.