Immunolocalization of Steroidogenic Enzymes in Equine Fetal Adrenal Glands During Mid-Late Gestation

Abstract The concentrations of inhibin and follistatin in amniotic fluid and in tissue extracts from the placenta, gonads and adrenals of fetal sheep were measured using radioimmunoassays. These tissue extracts were from whole fetuses from days 16 to 45 and from the individual organs from day 46 to 145 (term) and were assayed at multiple dilutions. The capacity of these extracts to alter FSH production of rat anterior pituitary cells in culture was also assessed at multiple dilutions. Immunoactive inhibin concentrations in amniotic fluid from both sexes increased during gestation and levels were significantly greater in males than females. Peak concentrations of immunoreactive inhibin of 11·2±1·9 ng/ml were found in males at 116–125 days of gestation. Follistatin concentrations did not change throughout gestation and no significant difference was noted between sexes. Mean follistatin levels throughout gestation were 3·0±0·9 ng/ml for males and 3·7±0·9 ng/ml for females. Despite the potential for FSH inhibition by inhibin and follistatin, amniotic fluid from both sexes at all stages of gestation stimulated FSH secretion in the pituitary cell bioassays, suggesting the presence of activin which was confirmed by the measurement of immunoactive activin (13·3±2·5 ng/ml) in a specific radioimmunoassay. Maximum concentrations of immunoactive and bioactive inhibin in placental extracts were observed in late gestation (2·2 ±0·6 and 3·8±1·6 ng/g respectively) and there was no significant difference between sexes. Follistatin concentrations in placental cotyledons ranged from 11·5 to 27·1 ng/g with no significant difference between sexes. In view of the higher follistatin concentrations compared with inhibin, it is likely that the capacity of placental extracts to suppress FSH production by pituitary cells in culture is due predominantly to follistatin. Immunoactive inhibin was observed in high concentrations in the fetal testis throughout gestation; with concentrations increasing to a maximum of 1993·0± 519·7 ng/g at 126–135 days of gestation with a ratio of bioactive: immunoactive inhibin of 1:20. Although bioactive and immunoactive inhibin was also observed in fetal ovaries and adrenals from both male and female fetuses, concentrations were lower than those observed in fetal testes. Follistatin concentrations in the fetal testis were elevated between 70 and 95 days (97·6 ng/g) and then declined. Similar concentrations were found in the adrenal glands of both sexes (males 83·5–103·3 ng/g: females 55·3–95·8 ng/g). In both males and females, immunoactive inhibin concentrations in fetal adrenals increased during gestation peaking at levels of 34·4±16·5 and 27·8± 9·0 ng/g respectively. These data suggest that the capacity of adrenal extracts to suppress FSH production by pituitary cells is due to both inhibin and follistatin. These studies demonstrated that significant concentrations of immunoactive inhibin and follistatin are present in amniotic fluid, and the fetal gonads, adrenal glands and placenta in sheep. The role of these proteins during fetal development requires further study. Journal of Endocrinology (1994) 141, 219–229

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The expression of ovine placental lactogen, StAR and progesterone-associated steroidogenic enzymes in placentae of overnourished growing adolescent ewes

Reproduction ◽

10.1530/rep-06-0294 ◽

2007 ◽

Vol 133 (4) ◽

pp. 785-796 ◽

Cited By ~ 25

Author(s):

Richard G Lea ◽

Peter Wooding ◽

Ian Stewart ◽

Lisa T Hannah ◽

Stephen Morton ◽

...

Keyword(s):

Intrauterine Growth Restriction ◽

Nutrient Intake ◽

Hepatic Clearance ◽

Transcript Abundance ◽

Late Gestation ◽

Placental Lactogen ◽

Steroidogenic Enzymes ◽

Intrauterine Growth ◽

Pregnant Adolescent ◽

Singleton Pregnancies

Overnourishing pregnant adolescent sheep promotes maternal growth but reduces placental mass, lamb birth weight and circulating progesterone. This study aimed to determine whether altered progesterone reflected transcript abundance forStAR(cholesterol transporter) and the steroidogenic enzymes (Cyp11A1,Hsd3bandCyp17). Circulating and placental expression of ovine placental lactogen (oPL) was also investigated. Adolescent ewes with singleton pregnancies were fed high (H) or moderate (M) nutrient intake diets to restrict or support placental growth. Experiment 1: peripheral progesterone and oPL concentrations were measured in H (n=7) and M (n=6) animals across gestation (days 7–140). Experiment 2: progesterone was measured to mid- (day 81; M:n=11, H:n=13) or late gestation (day 130; M:n=21, H:n=22), placental oPL,StARand steroidogenic enzymes were measured by qPCR and oPL protein by immunohistochemistry. Experiment 1: in H vs M animals, term placental (P<0.05), total cotyledon (P<0.01) and foetal size (P<0.05) were reduced. Circulating oPL and progesterone were reduced at mid- (P<0.001,P<0.01) and late gestation (P<0.01,P<0.05) and oPL detection was delayed (P<0.01). Experiment 2: placental oPL was not altered by nutrition. In day 81 H animals, progesterone levels were reduced (P<0.001) but not related to placental or foetal size. Moreover, placental steroidogenic enzymes were unaffected. Day 130 progesterone (P<0.001) andCyp11A1(P<0.05) were reduced in H animals with intrauterine growth restriction (H+IUGR). Reduced mid-gestation peripheral oPL and progesterone may reflect altered placental differentiation and/or increased hepatic clearance respectively. Restricted placental growth and reduced biosynthesis may account for reduced progesterone in day 130 H+IUGR ewes.

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Changes in blood flow distribution during the perinatal period in fetal sheep and lambs

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y92-226 ◽

1992 ◽

Vol 70 (12) ◽

pp. 1576-1582 ◽

Cited By ~ 15

Author(s):

Michelle P. Bendeck ◽

B. Lowell Langille

Keyword(s):

Blood Flow ◽

Adrenal Glands ◽

Flow Distribution ◽

Perinatal Period ◽

Tissue Growth ◽

Late Gestation ◽

Blood Flow Distribution ◽

Total Blood ◽

Fetal Sheep ◽

Blood Flows

We have measured total blood flows and blood flows per 100 g tissue to major tissues at 120 and 140 days gestation in fetal sheep and at 3 and 21 days of age in lambs (gestation period = 144 ± 2 days). Between 120 and 140 days gestation, flow per 100 g tissue increased by 74, 150, and 317% in the renal, intestinal, and hepatic arterial beds, but no further significant change in flow was observed at 3 or 21 days postpartum. Blood flows per 100 g to cerebral hemispheres and cerebellar tissues also increased dramatically during late gestation (142 and 121%, respectively), but declined sharply by 3 days postpartum (73 and 75%, respectively). Brain blood flows at 21 days postpartum remained substantially below late gestational levels. Adrenal blood flows per 100 g more than doubled during late gestation, fell by more than half at birth, and only partially recovered by 21 days of age. Blood flows to carcass tissues did not change in late gestation, fell at birth, then partially recovered. Pre- and post-natal increases in brain blood flows were almost entirely attributable to increased perfusion rather than tissue growth, whereas large perinatal increases in flow to the diaphragm paralleled tissue growth. Tissue growth and increased perfusion per 100 g contributed almost equally to increased blood flows to kidneys postnatally, and to adrenal glands and the gastrointestinal tract prenatally.Key words: blood flow, perinatal, birth, fetus, sheep.

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Expression of steroidogenic enzymes in the bovine placenta and fetal adrenal glands throughout gestation.

Endocrinology ◽

10.1210/endo.130.5.1374010 ◽

1992 ◽

Vol 130 (5) ◽

pp. 2641-2650 ◽

Cited By ~ 18

Author(s):

A J Conley ◽

J R Head ◽

D T Stirling ◽

J I Mason

Keyword(s):

Adrenal Glands ◽

Steroidogenic Enzymes ◽

Bovine Placenta

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Androgenic potential of human fetal adrenals at the end of the first trimester

Endocrine Connections ◽

10.1530/ec-17-0085 ◽

2017 ◽

Vol 6 (6) ◽

pp. 348-359 ◽

Cited By ~ 7

Author(s):

I Savchuk ◽

M L Morvan ◽

J P Antignac ◽

K Gemzell-Danielsson ◽

B Le Bizec ◽

...

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Transcription Factors ◽

Western Blotting ◽

Adrenal Glands ◽

First Trimester ◽

Gas Chromatography Mass Spectrometry ◽

Steroidogenic Enzymes ◽

Complex Process ◽

Low Expression

The onset of steroidogenesis in human fetal adrenal glands (HFA) during the first trimester is poorly investigated. An unresolved question is the capacity of the HFA to produce potent androgen DHT via conventional and/or the backdoor pathway(s) at the end of first trimester, when androgen-responsive organs are developed. Our aim was to explore steroidogenesis and the expression of steroidogenic enzymes and transcription factors in HFA at gestational weeks (GW) 9–12 with focus on their androgenic potential. Steroids in the HFA were analyzed by gas chromatography/mass spectrometry. The expression of steroidogenic enzymes and transcription factors in the HFA at GW9–12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that during GW9–12 HFA produced steroids of the ∆5, ∆4 and the backdoor pathways of the biosynthesis of DHT, though the latter was limited to production of 17α-OH-dihydroprogesterone, androsterone and androstanedione without further conversion to DHT. The only androgens identified in the HFA were testosterone and androsterone, a precursor in the biosynthesis of DHT. We also observed higher levels of CYP17A1 but low expression of 3βHSD2 at GW11–12 in the HFA. Elevated levels of CYP17A1 were associated with an increased expression of SF-1 and GATA-6. Altogether, our data demonstrate that of those steroids analyzed, the only potent androgen directly produced by the HFA at GW9–12 was testosterone. The onset of steroidogenesis in the HFA is a complex process that is regulated by the coordinated action of related transcription factors.

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The effect of fetal hypophysectomy with or without ACTH replacement on the molecular weight profile of enkephalin-containing peptides in the adrenal medulla of the fetal sheep

Journal of Endocrinology ◽

10.1677/joe.0.1340369 ◽

1992 ◽

Vol 134 (3) ◽

pp. 369-375 ◽

Cited By ~ 8

Author(s):

C. L. Coulter ◽

I. R. Young ◽

C. A. Browne ◽

I. C. McMillen

Keyword(s):

Molecular Weight ◽

Adrenal Glands ◽

Enzymatic Digestion ◽

Late Gestation ◽

Fetal Sheep ◽

Carboxypeptidase B ◽

Significant Difference ◽

Group 5 ◽

Pregnant Ewe

ABSTRACT We have investigated the possible role of the fetal pituitary and ACTH in the control of the synthesis and post-translational processing of the enkephalin precursor, proenkephalin A (proEnk A), in the fetal sheep adrenal gland in late gestation. Fetal hypophysectomy (n = 8) or sham operations (n = 4) were performed between 109 and 118 days of gestation. At 138–139 days, either ACTH(1–24) (10·5 μg/0·24 ml saline per h, n = 4) was infused intravenously for 72 h into hypophysectomized fetal sheep or 0·9% (w/v) NaCl alone (0·24 ml/h, n = 4) was infused for 72 h into hypophysectomized fetal sheep and sham-operated animals. At the end of the infusion the pregnant ewe was killed and left or right adrenal glands (n = 12) were collected from the fetal sheep that were intact and given saline (Intact + sal; n = 4), hypophysectomized and given saline (Hx + sal; n = 4) and hypophysectomized and given ACTH (Hx + ACTH; n = 4). Each adrenal was homogenized in acid (acetic acid (1 mol/l)/HCl (20 mmol/l)/2-mercaptoethanol (0·2%)). After centrifugation, the supernatant was loaded onto a Sephadex G-75 column (2·0 × 50 cm), eluted at 80 ml/24 h and fractions were collected (5 ml, n = 42). An aliquot of each fraction (2 ml) was dried down prior to enzymatic digestion (trypsin/carboxypeptidase B) and oxidation with H2O2, and assay for methionine-O-enkephalin (immunoreactive Met-O-Enk). The total adrenal content of immunoreactive Met-O-Enk was significantly greater in the Hx + ACTH group (326·2 ±66·7 (s.e.m.)ng/adrenal) when compared with either the Intact + sal group (152·7 ±44·0 ng/adrenal) or the Hx + sal group (112·1 ±20·8 ng/adrenal). In the adrenal glands from all fetuses immunoreactive Met-O-Enk was found in four molecular weight ranges: < 12 kDa, 12–7 kDa, 7–3 kDa and < 3 kDa. There was no significant difference between the Hx + sal and Hx + ACTH groups in the proportion of immunoreactive Met-O-Enk present in each of the molecular weight ranges in the adrenals and therefore the data from these groups were combined for further statistical analysis. The proportion of immunoreactive Met-O-Enk in the > 12 kDa range was significantly less in the Intact + sal group (5·5 ±2·3%) when compared with the hypophysectomized sheep with or without ACTH replacement (18·7 ± 4·5%). These data demonstrate that fetal hypophysectomy alters the molecular weight profile of Enk-containing peptides in the adrenal of the fetal sheep and whilst ACTH replacement in the hypophysectomized fetus does not alter the post-translational processing of the Enk-containing peptides, it stimulates an increase in the total amount of immunoreactive Met-O-Enk in the fetal adrenal in late gestation. Journal of Endocrinology (1992) 134, 369–375

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Hypocretin/orexin increases the expression of steroidogenic enzymes in human adrenocortical NCI H295R cells

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.91034.2008 ◽

2009 ◽

Vol 297 (5) ◽

pp. R1601-R1609 ◽

Cited By ~ 28

Author(s):

Jan Wenzel ◽

Nicole Grabinski ◽

Cordula A. Knopp ◽

Andreas Dendorfer ◽

Manjunath Ramanjaneya ◽

...

Keyword(s):

Adrenal Glands ◽

Synthesis Rate ◽

Receptor Subtypes ◽

Mrna Levels ◽

Steroidogenic Enzymes ◽

Orexin A ◽

Orexin Receptors ◽

Adrenal Steroidogenesis ◽

Intracellular Ca ◽

H295r Cells

Hypocretins/orexins act through two receptor subtypes: OX1 and OX2. Outside the brain, orexin receptors are expressed in adrenal glands, where orexins stimulate the release of glucocorticoids. To further address the regulation of steroidogenesis, we analyzed the effect of orexins on the expression of steroidogenic enzymes in human adrenocortical National Cancer Institute (NCI) H295R cells by qPCR. In NCI H295R cells, OX2 receptors were highly expressed, as they were in human adrenal glands. After treatment of NCI H295R cells with orexin A for 12–24 h, the cortisol synthesis rate was significantly increased, whereas 30 min of treatment showed no effect. While CYP11B1 and CYP11B2 mRNA levels were increased already at earlier time points, the expression of HSD3B2 and CYP21 mRNA was significantly up-regulated after treatment with orexin A for 12 h. Likewise, orexin B increased CYP21 and HSD3B2 mRNA levels showing, however, a lower potency compared with orexin A. The mRNA levels of CYP11A and CYP17 were unaffected by orexin A. OX2 receptor mRNA levels were down-regulated after 12 and 24 h of orexin A treatment. Orexin A increased intracellular Ca2+ but not cAMP concentrations in NCI H295R cells. Furthermore, inhibition of PKC and MAPK kinase/ERK kinase (MEK1/2) prevented the increase of HSD3B2 expression by orexin A. Accordingly, orexin A treatment of NCI H295R cells markedly enhanced ERK1/2 phosphorylation that was prevented by PKC and, in part, PKA inhibition. In conclusion, orexins may influence adrenal steroidogenesis by differential regulation of the expression of steroidogenic enzymes involving Ca2+, as well as PKC-ERK1/2 signaling.

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Forage allowances offered to pregnant ewes until middle and late gestation: Organ priorities on foetus development

South African Journal of Animal Science ◽

10.4314/sajas.v50i3.14 ◽

2020 ◽

Vol 50 (3) ◽

pp. 471-480

Author(s):

M. J. Abud ◽

A. Bielli ◽

J. Ithurralde ◽

A. Freitas-de-Melo ◽

A. Álvarez-Oxiley ◽

...

Keyword(s):

Intrauterine Growth Restriction ◽

Adrenal Glands ◽

External Genitalia ◽

Liver Weight ◽

Reproductive Organ ◽

Late Gestation ◽

Organ Weights ◽

Intrauterine Growth ◽

The Central Nervous System ◽

High Forage

Effect of forage allowance before conception and until mid or late gestation was evaluated for effects on foetal and neonatal weights, carcass, nervous systems, metabolic and reproductive organ weights, body dimensions, and variation in intensity of the effects among organs. Effects of two forage allowances, HFA: high forage allowance (2.9 - 3.8 kg of dry matter (DM)/kg bodyweight (BW)) and LFA: low forage allowance (1.4 - 2.6 kg DM/kg BW) were evaluated from 23 days before conception until 70 or 122 days postpartum. On gestation day 70, nine ewes per treatment, each carrying one male foetus, were euthanized and their foetuses were removed. The foetuses were weighed, their carcass and organ weights were recorded, and their external genitalia dimensions were measured. Nine additional lambs per treatment were euthanized 12 hours after birth and the same data were recorded. Hearts from day 70 LFA foetuses were lighter, their external genitalia were smaller, and their foetal weight tended to be less than in HFA. Newborn lambs from LFA ewes had lighter carcasses, livers, kidneys, adrenal glands and testes, shorter penises, but higher brain to liver weight ratios than in HFA. The cerebellum, brain, and heart weights of LFA and HFA newborn lambs did not differ. Low forage allowance until late gestation influenced both foetal and lamb weights and affected organ weights differentially. Thus, the treatments induced differences in prioritization of nutrients, with the central nervous system receiving the highest priority, and carcass and external genitalia the lowest. Keywords: foetal programming, intrauterine growth restriction, lambs, undernutrition

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