Abstract
Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3β-hydroxysteroid dehydrogenase (3βHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3βHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3βHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3βHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.
Introduction
The establishment and maintenance of pregnancy, and the initiation of parturition, have long been recognized to rely on hormones including progestins, estrogens and prostaglandins, the secretion of which are suppressed or promoted dependent on stage of gestation (Thorburn et al. 1977). In cattle, we and others have focused on progesterone and estrogens (estrone, estradiol and estrone sulphate) as the major steroids involved, placental synthesis and target tissues (Conley & Ford 1987, Hoffmann & Schuler 2002, Schuler et al. 2008, Nguyen et al. 2012, Conley & Reynolds 2014, Schuler et al. 2018), along with placental enzyme expression (Conley et al. 1992, Schuler et al. 2006). However, recent studies in our laboratory investigating steroid secretion during equine pregnancy have identified 5α-reduced pregnanes, 5α-dihydroprogesterone (DHP) specifically, as an important progestin (Scholtz et al. 2014, Legacki et al. 2016, 2017, 2018, Reynolds et al. 2018) activating the progesterone receptor in a species-specific manner (Scholtz et al. 2014). Other direct metabolites of DHP exhibit other biopotent effects. As potent neuro-active steroids, 5α-reduced pregnanes, such as allopregnanolone (5α-pregnan-3α-ol-20-one or 3αDHP), have been hypothesized to suppress activation of the fetal pituitary–adrenal axis until parturition (Conley & Neto 2008, Brunton et al. 2014, Conley & Reynolds 2014). Measuring 5α-reduced pregnanes by immuno-assay is difficult and requires mass spectrometry with appropriate method development (Legacki et al. 2016), an approach that has been little utilized in ruminant species to date. To the authors’ knowledge, only one study has applied mass spectrometry to monitor changes in steroid hormone concentrations during gestation and parturition in cattle (Martins-Júnior et al. 2014), but 5α-reduced steroids were not included in that analysis. Thus, the studies examining changes in hormone concentrations in late pregnancy and at parturition in cattle have to date investigated only a limited number of steroids. None, to our knowledge, have examined 5α-reduced pregnane concentrations during gestation in cattle.
The role of fetal adrenal activation and cortisol secretion in fetal preparation for (Silver 1990), and the initiation of (Thorburn & Challis 1979), parturition are well known and have been investigated extensively in sheep in particular (Challis et al. 2000). Notwithstanding the significant contribution of the corpus luteum to pregnancy maintenance even in the final weeks of bovine gestation, a similar sequence of events is hypothesized to initiate parturition in cattle (Comline et al. 1974). The administration of synthetic corticoids, like dexamethasone (DEX), induces parturition in late gestation and is thought to trigger events in a way that resembles the spontaneous, natural birth process initiated by fetal adrenal activation (Adams & Wagner 1970). Recent, comprehensive studies (Shenavai et al. 2012) have compared many facets of placental gene expression and steroid secretion in cows induced to calve using DEX administration as well as a progesterone receptor antagonist, prostaglandin F2α, and natural spontaneous birth. Others studying DEX-induced parturition in cattle (Adams & Wagner 1970, Comline et al. 1974, Hirayama et al. 2012) have focused on the effects on the placenta (Hoffmann & Schuler 2002) with less attention given to luteal function. The current studies were conducted to re-evaluate DEX-induced parturition in cows in late gestation (day 270) by examining (1) the array of pregnenes (Δ5, pregnenolone; Δ4, progesterone and metabolites) and pregnanes (DHP and metabolites) in serum and in tissues, (2) the expression of enzymes (both transcript abundance and catalytic activities) involved in placental progesterone synthesis, as well as (3) luteal tissue progesterone concentrations and 3βHSD enzyme activity. It was hypothesized that DEX treatment would alter placental pregnene and pregnane synthesis and thereby facilitate progesterone withdrawal in the late gestation cow.
Materials and methods
Animals, treatments and sample collection
Experiments were approved by the Institutional Animal Use and Care Advisory Committee at the University of California, Davis and North Dakota State University (NDSU), and were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Cows were synchronized as previously described (Larson et al. 2006) and pregnancies established (day 0) by artificial insemination with frozen semen from a single sire. At gestational day 270, cows were randomly assigned to one of two treatments. Cows were either injected with saline alone (SAL, 4 mL; n = 8) or DEX (dexamethasone sodium phosphate) suspended in saline at 10 mg/mL (4 mL; n = 9). Blood samples were taken by jugular venipuncture just before treatment and at slaughter and allowed to clot. Serum was harvested and stored at −20°C. Thirty-eight hours after treatment, cows were slaughtered at the NDSU Meats Laboratory, a federally inspected facility, using approved methodology (AVMA 2013). At slaughter, the entire reproductive tract was obtained, and placenta (caruncle (maternal placental tissue) and cotyledon (fetal placental tissue)) and corpus luteum were dissected. Samples of caruncle, cotyledon and corpus luteum were snap-frozen in liquid nitrogen-cooled isopentane as described previously (Reynolds et al. 2015, 2018) and were stored at −80°C until they were used for analysis of tissue steroid concentrations and steroidogenic activities.
Microsomal and mitochondrial enrichment
Placental tissues were homogenized in buffer (0.1 M K3PO4 pH 7.4, 20% glycerol, 5 mM β-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride and 1 μg/μL aprotinin) and then briefly sonicated. Homogenates were centrifuged at 15,000 g for 10 min to recover mitochondria and the supernatant was transferred to a new tube and centrifuged again at 100,000 g for 1 h to recover microsomes, as previously described and validated (Moran et al. 2002, Corbin et al. 2016, Legacki et al. 2017, 2018, Reynolds et al. 2018). The resulting pellets (15,000 g – mitochondrial; 100,000 g – microsomal) were recovered and resuspended. The concentrations of crude protein in each were determined using the Pierce BCA Protein Reagent (Thermo Scientific). Microsomal preparations were stored in aliquots at −80°C.
Standards and solutions
Standards were purchased from Steraloids (Newport, RI): 5α-dihydroprogesterone (5α-pregnan-3,20-dione, DHP), allopregnanolone (5α-pregnan-3α-ol-20-one or 3αDHP), 5α-pregnan-3β, 20α-diol (3β,20αDHP), 5α- pregnan-20α-ol-3-one (20αDHP), pregnenolone, progesterone, 20αOH-progesterone, androstenedione (A4), testosterone dehydroepiandrosterone (DHEA), d7-androstenedione (A4-d7), d3-testosterone (T-d3) and d9-progesterone (P4-d9). A master mix of all reference standards was prepared and diluted in methanol (10, 1, 0.1 and 0.01 ng/mL). Methanol and water were of HPLC grade and obtained from Burdick and Jackson (Muskegon, MI, USA). Formic acid and methyl-tert butyl ether were of ACS grade and obtained from EMD (Gibbstown, NJ, USA).
Enzyme activities
Both 3βHSD and 5α-reductase enzyme activities were examined in sub-cellular fractions of placental tissues. The activity of 3βHSD was determined as described earlier (Conley et al. 2012), with minor modifications. Cotyledonary 3βHSD activity was 4-fold higher than that in caruncular samples (data not shown) and thus subsequent studies focused on microsomal and mitochondrial sub-cellular fractions from cotyledonary tissue samples. Luteal tissues were treated similarly, but reactions utilized less protein incubated for shorter periods because of the high reaction rates relative to placental samples. Accordingly, reactions were conducted with 10–20 μg of microsomal or mitochondrial protein in phosphate buffer (100 mM K3PO4, 1 mM EDTA pH 7.4) with pregnenolone (10 μM final) as substrate at 37°C for 0.25–2 h and 10 mM βNAD. The incubation time was chosen, based on preliminary time course experiments with sample pools, to ensure that substrate was not limiting. Parallel reactions were conducted with addition of trilostane (100 μM), a specific inhibitor of 3βHSD, to verify that the steroids detected after incubation resulted from active synthesis. In addition, 5α-reductase activity was assessed in microsomal incubations conducted with 3 μM progesterone with and without finasteride (100 μM; specific 5α-reductase inhibitor) to confirm active synthesis, essentially as described previously for equine epididymis (Corbin et al. 2016). These reactions were supported by co-incubation with NADPH and a generating system (1 mM NADP+, 10 mM glucose-6-phosphate and 1.25U glucose-6-phosphate dehydrogenase).
Steroid product analysis
The LC-MS/MS method used has been validated as described previously for serum/plasma (Legacki et al. 2016) and tissue steroids (Legacki et al. 2017). This method detects progesterone, pregnenolone, DHEA, A4, DHP and several of its metabolites including 3αDHP (allopregnanolone), 20αDHP, 3β,20αDHP. Briefly, d7-A4 and d9-P4 internal standards were added to all serum samples which were extracted subsequently with methyl-tert butyl ether (1:5). The products of reactions for 5α-reductase and 3βHSD were extracted and prepared similarly. Frozen tissues were ground mechanically (Precellys, Rockville, MD, USA) using stainless steel beads (3.2 mm), extracted with a 50:50 mix of methanol:water and shaken at 6500 rpm for 60 s. Samples were then stored for 20 min at −20°C and shaken again using the same parameters. The supernatant was removed, put into glass screw top tubes and dried to completion. One milliliter of water was added to the dried homogenate, followed by the addition of 100 µL of the internal standard mixture (A4-d7, T-d3 and P4-d9) in methanol. Calibrators ranged from 0.1 to 100 ng/mL and four levels of quality controls (QC; 0.6, 1.5. 20 and 80 ng/mL) were prepared alongside the samples, transferred into 12 × 75 glass tubes and dried in a Zymark Turbovap Concentrator (Hopkinton, MA, USA) at 45°C under N2. Samples were reconstituted with 200 µL of 50:50 mix of water and methanol. The reverse-phase gradient separation was performed on an Agilent UHPLC C18 analytical column (2.1 × 50 mm, 1.8 µm ps) with two mobile phases delivered at 0.4 mL/min, an injection volume of 20 µL and a column temperature of 40°C. Mobile phase A and B were water with 0.2% formic acid and methanol, respectively. An elution gradient was held at 40% B for the first 0.2 min, 40–60% B from 0.2 to 1 min, 60–80% B from 1 to 10 min, 80–90% B from 10.0 to 10.1 min, held at 90% B from 10.1 to 11.1 min, 90%–40% from 11.1 to 11.2 min and held at 40% B until 13.10 min. Ionization achieved utilized an atmospheric-pressure chemical ionization (APCI) source. Tandem mass spectral detection was accomplished using a Bruker EVOQ (Bruker Daltronics Inc., Billerica, MA, USA). Detection and quantitation of all analytes were accomplished using multiple reaction monitoring with a minimum of two transitions per analyte. Inter- and intra-accuracy and precision were assessed at four QC concentrations for all analytes (six replicates). All analytes were measured with ≤15% deviation from expected concentrations for the three highest QC concentrations (1.5, 20 and 80 ng/mL) and ≤15% coefficient of variation (%CV). For the lowest QC concentration (0.6 ng/mL) pregnenolone, 20αDHP and 3β,20αDHP had ≤20% deviation from expected concentrations. All analytes measured had a percent accuracy (%Acc) >90% and a precision <15%. The responses for all analytes were linear and gave correlation coefficients (R2) of >0.99.
Transcript abundance
Expression levels of transcripts encoding steroid synthesizing and metabolizing enzymes were determined by quantitative reverse-transcriptase/polymerase chain reaction (qPCR) analysis using SYBR green chemistry and the ΔΔCT method (Livak & Schmittgen 2001) using primers as previously described (Reynolds et al. 2015, 2018). All samples were compared to a reference pool comprising RNA isolated from bovine caruncles (maternal portions of the placenta) and fetal adrenal glands from the SAL-treated cows from this study. In addition, expression of β-actin was used as the endogenous reference.
Statistical analysis
The differences in concentration among each of the measurable steroids in serum, tissue and products of steroidogenic enzyme assays were subjected to ANOVA using the Proc Mixed function in SAS (SAS Statistical Software, SAS Institute Inc.). Analysis of serum steroid concentrations before and after treatment incorporated a repeated-measures design. Main effects of treatment, and for serum data treatment by time interactions, were determined and Pearson’s correlation co-efficients were determined. Differences among means were examined by orthogonal contrasts. The data were graphed using the means and standard errors. Expression of select transcripts was analyzed as the ΔCT by subtracting the CT of the ACTB from the CT of the transcript of interest (Livak & Schmittgen 2001). Expression data are presented as median and range for ΔCT.
Results
Serum steroid concentrations
Progesterone was present at concentrations ranging from 3 to 5 ng/mL before treatment (Fig. 1). Progesterone concentrations did not change with time in the SAL injected cows but were lower in cows after DEX treatment with significant main effects of treatment and time (P < 0.05) and a significant treatment by time interaction (P < 0.01). Luteal weight also tended to be lower in DEX- than in SAL-treated cows (5.24 ± 0.30 vs 5.82 ± 0.25 g, P < 0.08) (Supplementary Figure 1 (see section on Supplementary data given at the end of the article)). Several other 5α-reduced pregnanes and pregnenes were detected included allopregnanolone (3αDHP), pregnenolone (P < 0.05), DHP and 20αOH-progesterone, all of which were <1 ng/mL on average (Fig. 1). Dexamethasone treatment led to a significant decrease in serum concentrations of pregnenolone, DHP (P < 0.05) and allopregnanolone at the time of slaughter (P < 0.01; Fig. 1). Notably also, 20αOH-DHP was by far the most abundant steroid measured, present in some cows at >100 ng/ml (Fig. 2), more than 20-fold higher than progesterone and over 100-fold higher than the other measured 5α-reduced pregnanes before treatment. There was a decrease in 20αDHP with time (P < 0.05), but no main effects of DEX and no interaction.
Serum steroid concentrations (ng/mL) in saline- (SAL) and dexamethasone-treated (DEX) cows before and 38 h after treatment on day 270 of gestation. Steroids concentrations were determined by LC-MS/MS, as described in Materials & methods. For each steroid depicted, differences between means are indicated, a,bP < 0.05, a,cP < 0.01.
Citation: Reproduction 157, 5; 10.1530/REP-18-0558
Serum concentrations (ng/mL) of 20α-hydroxy-5α-dihydroprogesterone (20αOH-DHP) in saline- (SAL) and dexamethasone-treated (DEX) cows before and 38 h after treatment on day 270 of gestation. Steroids concentrations were determined by LC-MS/MS, as described in Materials & Methods. a,bMeans with different superscripts differ, P < 0.05.
Citation: Reproduction 157, 5; 10.1530/REP-18-0558
Tissue steroid concentrations
The cotyledonary (fetal) component of the placentome had significantly higher tissue concentrations of pregnenes (pregnenolone and progesterone; P < 0.01) and pregnanes (DHP and allopregnanolone; P < 0.05) than the caruncular (maternal) component of the placentomes (Fig. 3). Pregnenolone was the highest of the measured steroids in the cotyledon (182.3 ± 51.5 ng/g tissue) and progesterone (90.4 ± 30.0 ng/g tissue) the next highest. Pregnenolone was also the steroid in highest concentrations in the caruncle (47.6 ± 7.3 ng/g tissue). Treatment with DEX reduced progesterone in cotyledons (93.44 ± 30.53 vs 38.16 ± 8.44 ng/g tissue, P < 0.05) and allopregnanolone in caruncles (7.29 ± 1.72 vs 0.88 ± 0.88 ng/g, P < 0.05), but did not affect cotyledonary pregnenolone or DHP (Fig. 3). There was no correlation between cotyledonary and serum concentrations of progesterone.
Steroid concentrations (ng/g) in cotyledonary and caruncular tissues from saline- (SAL) and dexamethasone-treated (DEX) cows collected at slaughter 38 h after treatment on day 270 of gestation. Steroids concentrations were determined by LC-MS/MS, as described in Materials & Methods. For each steroid depicted, differences between means are indicated, a,bP < 0.01, a,cP < 0.05, b,cP < 0.05.
Citation: Reproduction 157, 5; 10.1530/REP-18-0558
Steroid concentrations in luteal tissue (Fig. 4) were, on average, far higher than those in placental tissues. Luteal tissue progesterone concentrations (33.4 ± 1.9 μg/g) were 10-fold higher (P < 0.001) than any other steroid detected in the SAL-treated cows (pregnenolone, 3.2 ± 0.2 μg/g; DHP, 1.9 ± 0.2 μg/g; all others were <1 μg/mL; 20αOH-DHP was undetectable). Luteal progesterone concentrations in SAL-treated cows were significantly higher than those in luteal tissue from DEX-treated cows (33.4 ± 1.9 vs 19.1 ± 2.3 μg/g, respectively; Fig. 4; P < 0.05). In DEX-treated cows, concentrations of progesterone in luteal tissue were positively correlated with those in serum over all (r = +0.69, P < 0.05) even including what appeared to be a single outlying data point. If this single data point was removed from the analysis, the correlation co-efficient increased markedly (r = +0.98, P < 0.0001). In SAL-treated cows, there was no significant correlation between serum progesterone and luteal tissue progesterone concentrations, but luteal progesterone was negatively correlated with serum pregnenolone (r = −0.81, P < 0.05).
Steroid concentrations (ng/g) in corpus luteum tissues from saline- (SAL) and dexamethasone-treated (DEX) cows collected at slaughter 38 h after treatment on day 270 of gestation. Steroids concentrations were determined by LC-MS/MS, as described in Materials & methods. For each steroid depicted, differences between means are indicated, a,bP < 0.05.
Citation: Reproduction 157, 5; 10.1530/REP-18-0558
Enzyme activities
The activity of 3βHSD in cotyledonary microsomes was on average four-fold higher than caruncular samples and trilostane effectively inhibited >90% of product accumulation (data not shown). Cotyledonary microsomal and mitochondrial fractions were examined further (Table 1). The differences in rate of conversion of pregnenolone to progesterone between cotyledonary microsomal and mitochondrial proteins from SAL- and DEX-treated cows were not large relative to the mean activities themselves and not greatly affected by DEX (range of mean rates, 6.02–7.26 nmol/mg/h; Table 1). There was a significant main effect of sub-cellular fraction on 3βHSD activity (P < 0.01) and a significant interaction (P < 0.05) between treatment and sub-cellular fraction (P < 0.05). Cotyledonary mitochondrial 3βHSD was significantly higher in DEX than SAL-treated cows (P < 0.01). On average, however, there was 2.35-fold more total microsomal than mitochondrial protein isolated from cotyledonary tissues, which was similar in tissues from SAL-treated and DEX-treated cows (microsomal/mitochondrial protein, 2.44- vs 2.27-fold, respectively). Overall there was far more 3βHSD activity in luteal than cotyledonary microsomes and mitochondria (Table 1). Within luteal tissue there was a main effect of sub-cellular fraction, with significantly more activity in microsomes than mitochondria (P < 0.001). However, there was no main effect of DEX treatment and no interaction, only a tendency for higher luteal 3βHSD activity in mitochondria from DEX- than from SAL-treated cows (P < 0.07).
Activity (nmol/mg/h) of 3β-hydroxysteroid dehydrogenase in microsomal and mitochondrial protein isolated from cotyledonary and corpus luteum tissue from saline- (SAL; n = 8) and dexamethasone- (DEX; n = 9) treated cows in late gestation (270 days), 38 h after treatment.
| Cotyledon | Corpus luteum | |||
|---|---|---|---|---|
| Microsomal | Mitochondrial | Microsomal | Mitochondrial | |
| SAL | 6.45 ± 0.07a | 6.69 ± 0.06a | 42.79 ± 0.67a | 26.72 ± 0.87b |
| DEX | 6.02 ± 0.05a | 7.26 ± 0.07b | 43.05 ± 0.66a | 32.15 ± 0.32b |
Shown are the means ± standards errors of the means.
a,bWithin cotyledon and corpus luteum, means with different superscripts differ, P < 0.01.
Estimates of 5α-reductase activity in microsomal protein were based on the rate of accumulation of DHP. Caruncular tissues synthesized DHP at a rate of 2.02 ± 0.24 nmol/mg/h and 2.15 ± 0.29 nmol/mg/h in SAL- and DEX-treated animals, respectively, which was significantly greater than in cotyledonary tissues (P < 0.05). Cotyledonary tissues synthesized DHP at a rate of 1.16 ± 0.20 and 1.47 ± 0.27 nmol/mg/h in SAL- and DEX-treated animals, respectively. Dexamethasone treatment did not affect the activity of 5α-reductase in either caruncular or cotyledonary tissue. Finasteride inhibited DHP synthesis, verifying active synthesis by 5α-reductase (data not shown).
Transcript analysis
The abundance of transcripts encoding enzymes and co-factors involved in steroid synthesis and metabolism in the placenta demonstrated the predominant steroidogenic role of the cotyledon over the caruncle. The abundance of StAR (P < 0.001), CYP11A1 (P < 0.0001) and its redox partner FDX (P < 0.05, though not FDXR, P > 0.3; Fig. 5) and of HSD3B1 (P < 0.001; Fig. 6) were all significantly higher in cotyledonary than in caruncular tissues. In contrast, the abundance of SRD5A1 was greater (P < 0.001) in caruncular than cotyledonary tissues. Of the placental steroidogenic enzyme transcripts investigated, the only detected effect of DEX was observed for SRD5A2 where DEX-treated cows had greater abundance (P < 0.001) in caruncular tissue than caruncular tissue of SAL-treated cows or cotyledonary tissue of either treatment (Fig. 6). Transcripts encoding CYP11A1, FDX, FDXR, StAR and HSD3B1 (Fig. 7) were more abundant in luteal than placental tissues from SAL- and DEX-treated cows (P < 0.01). In addition, transcript abundance was significantly lower in luteal tissue from DEX- than from SAL-treated cows for CYP11A1 (P < 0.005), as well as FDX, FDXR, StAR and HSD3B1 (P < 0.05).
Quantitative reverse transcriptase polymerase chain reaction (qPCR) analysis of transcript abundance in tissues collected at slaughter from cows treated 38 h earlier with dexamethasone (DEX) or saline (SAL). (A) CYP11A1 (encoding cytochrome P450 cholesterol side chain cleavage). (B) Ferrodoxin (FDX). (C) Ferrodoxin reductase (FDXR). (D) Steroid acute regulatory protein (StAR). Shown are the means and standard errors. a,bMeans with different superscripts differ, P < 0.05.
Citation: Reproduction 157, 5; 10.1530/REP-18-0558
Quantitative reverse transcriptase polymerase chain reaction (qPCR) analysis of transcript abundance in tissues collected at slaughter from cows treated 38 h earlier with dexamethasone (DEX) or saline (SAL). (A) HSD3B1 (encoding 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase). (B) SRD5A1 (5α-reductase type 1). (C) SRD5A2 (5α-reductase type 2). Shown are the means and standard errors. a,bMeans with different superscripts differ, P < 0.05.
Citation: Reproduction 157, 5; 10.1530/REP-18-0558
Quantitative reverse transcriptase polymerase chain reaction (qPCR) analysis of transcript abundance in luteal tissue collected at slaughter from cows treated 38 h earlier with dexamethasone (DEX) or saline (SAL). CYP11A1 (encoding cytochrome P450 cholesterol side chain cleavage), ferrodoxin (FDX), ferrodoxin reductase (FDXR), steroid acute regulatory protein (StAR) and HSD3B1. Shown are the means and standard errors. Means differ significantly as shown, *P < 0.05, **P < 0.005.
Citation: Reproduction 157, 5; 10.1530/REP-18-0558
Discussion
The results of this study provide new insight into the synthesis of pregnenes (progesterone, pregnenolone) and pregnanes (5α-reduced progesterone metabolites) in late pregnant cattle, both placental and luteal contributions to systemic levels, as well as the events triggered by DEX that induce bovine parturition. The 5α-reduced pregnane, 20α-DHP, was identified for the first time as an unusually abundant progesterone metabolite in these studies. To the best of our knowledge, this is the highest 5α-reduced pregnane ever identified in a ruminant. This suggests that 5α-reduction is a major route of progesterone metabolism in cattle, contrasting with the pregnant ewe wherein 5α-reduced pregnanes were typically less than 1 ng/mL in systemic blood (Reynolds et al. 2018). However, progesterone metabolism was not influenced by DEX, which successfully induced progesterone withdrawal, apparently by precipitating functional luteal regression based on the significantly lower luteal progesterone concentrations as well as transcripts encoding CYP11A1 and other enzymes and co-factors supporting progesterone synthesis in the corpora lutea of DEX-treated cows. The tendency for luteal weights to be less in DEX- compared with SAL-treated cows further suggests that structural regression had likely also begun, even though a significant decrease luteal weight might not be expected to be evident for 48 h (Juengel et al. 1993). These observations are consistent with results from an extensive series of experiments first reported in what the authors described as pilot studies on DEX induction of bovine parturition (Hoffmann et al. 1979). Thus, from the current data, it seems that DEX has no major effect on placental progesterone synthesis or metabolism (within 38 h) after administration (parturition reportedly occurs within 72 h after DEX; Adams & Wagner 1970, Shenavai et al. 2012) and that luteal regression precedes any subsequent, pre-parturient changes in placental steroidogenesis.
The relative contribution of the placenta to circulating progesterone concentrations in cows in late gestation was also clarified in the current studies by observations in the DEX-treated cows. Cotyledonary tissue concentrations of progesterone were not correlated with systemic progesterone concentrations, but luteal tissue concentrations were. The higher concentrations of pregnenolone and progesterone in cotyledonary than caruncular tissues was consistent with higher cotyledonary than caruncular 3βHSD enzyme activity and the higher transcript abundance of StAR, CYP11A1, FDX and HSD3B1 in the fetal component of the placenta. These data are consistent with a significant capacity for pregnene synthesis by the bovine placenta during gestation (Shemesh 1990, Conley et al. 1992), which may become relevant in the absence of a functional corpus luteum (Conley & Ford 1987). The extraordinarily high progesterone concentrations of luteal compared to cotyledonary tissue (>30,000 vs 100 ng/g) suggest that, despite the approximately 500-fold greater mass of cotyledons than of corpora lutea (Reynolds et al. 1990, Vonnahme et al. 2007), there is a more significant contribution of ovarian secretion to systemic progesterone concentrations in late gestation. The strong positive correlation between luteal tissue and systemic progesterone concentrations in DEX-treated cows is equally consistent and supportive evidence that luteal secretion is the major determinant of progesterone concentrations late in bovine gestation, as has been suggested by others (Schuler et al. 2008). Again, all the above data support the view that functional luteal regression more likely initiates progesterone withdrawal and parturition following DEX treatment in cattle.
Pregnenolone is the proximate precursor for the synthesis of progesterone, as well as all other pregnanes, and tissue concentrations are therefore relevant in terms of understanding substrate availability for steroidogenesis. Serum pregnenolone concentrations are reported here for the first time in pregnant cows, along with placental and in luteal tissue concentrations and 3βHSD enzyme activities. Cotyledonary pregnenolone concentrations were twice those of progesterone on average, despite what appears to be robust 3βHSD enzyme activity that were very similar in magnitude to previously published estimates in microsomes (Tsumagari et al. 1994). In fact, 3βHSD enzyme activity was as high in bovine cotyledon as it was in ovine cotyledon (Reynolds et al. 2018), which is adequate to sustain progesterone synthesis at levels compatible with maintenance of pregnancy in sheep. End product inhibition of placental 3βHSD activity by progesterone and 20αOH-progesterone is one potential reason why placental progesterone synthesis might not be maximized as long as luteal progesterone secretion dominates. Higher concentrations of pregnenolone than progesterone suggest again that substrate supply was not limiting in placental progesterone synthesis but 3βHSD may have been. In vitro cultured and perfused placentomal tissues recovered from cows after prostaglandin-induced luteolysis at 200 days of gestation showed increased progesterone synthesis from pregnenolone over tissues from SAL-treated cows (Conley & Ford 1987). Thus, excess pregnenolone coupled with the capacity of cotyledonary tissues to increase 3βHSD activity may provide the bovine placenta with the ability to compensate for the loss of luteal progesterone until late in gestation. Though 3βHSD activities were shown to be higher in luteal tissue by 5-fold over cotyledonary tissue, the difference in progesterone concentrations of several orders of magnitude suggest that cotyledonary 3βHSD activity may be limited not by the amount of enzyme but by other elements, perhaps reducing equivalents or the redox environment. This is also consistent with the lack of any demonstrated effect of DEX on the abundance of HSD3B1 or any of the transcripts encoding enzymes and associated proteins in placental tissues. Removal of the ovaries results in cows calving early in the absence of ovaries (Mcdonald et al. 1953, Estergreen et al. 1967) suggesting that, whatever contribution it might make, the capacity for placental ‘compensation’ wanes near term.
Some have concluded, primarily from in vitro studies, that bovine CL lose progesterone synthetic capacity as gestation proceeds (Mills & Morrissette 1970) and that the placenta plays an increasingly significant role supplementing luteal progesterone production as gestation advances (Shemesh 1990). Several researchers reported slight decreases in progesterone concentrations in luteal tissue in the second half of gestation (Melampy et al. 1959, Stormshak & Erb 1961, Bowerman & Melampy 1962, Erb et al. 1968) though correlations with ovarian venous concentrations were poor (Erb et al. 1968). Others have reported no difference in ovarian progesterone secretion between early and late gestation from in vitro perfusion studies (Mills & Morrissette 1970). A recent study, conducted to investigate concentrations of progesterone, pregnenolone and some 5α-reduced pregnanes in luteal tissue from cyclic cows using gas chromatography mass spectrometry, examined the effects of prostaglandin F2α-induced luteolysis (Waite et al. 2005). The response of the corpus luteum to prostaglandin F2α was rapid in these cyclic cows and was evident within 4 h of treatment. Despite a more protracted period between treatment and tissue collection, the results reported here for corpora lutea of late gestation bore a remarkable similarity in terms of the range of concentrations of progesterone and pregnenolone found in cyclic corpora lutea. There was slightly more progesterone and slightly less pregnenolone in the corpora lutea of pregnancy such that the progesterone:pregnenolone ratio was close to 10 compared with about 4 as reported for luteal tissue from cyclic cows (Waite et al. 2005). There were also 5α-reduced pregnanes at much lower concentrations in luteal tissues from late gestation cows including DHP (1–2 μg/g), which was not detected in corpora lutea of cyclic cows. Prostaglandin treatment of cyclic cows resulted in a decrease in luteal concentrations of progesterone, 20βOH-progesterone and pregnenolone and a significant increase in 3βOH DHP 24 h after treatment. In the current study, by comparison, while the decrease in luteal tissue pregnenolone was not significant, progesterone was significantly lower and the 3αOH DHP was significantly increased 38 h after DEX treatment. The tendency for lower luteal weight, coupled with the significant decreases in CYP11A1, FDX, FDXR, StAR and HSD3B1 transcript abundance, is consistent with functional regression, even though pregnenolone was not significantly affected by DEX treatment, and there was no demonstrated effect on 3βHSD activity. This might also reflect the delay between transcription, translation and protein processing needed to support steroidogenesis. Systemic concentrations of 20αOH DHP were already declining and were unaffected by DEX, so increased progesterone metabolism seems to be an equally unlikely explanation for the decrease in luteal progesterone. Changes in redox state of cells might have an influence on the availability of reducing equivalents needed to support 3βHSD in vivo (Sherbet et al. 2007) and, since these are supplied in vitro, an in vivo effect on the redox state of luteal cells could be easily missed. Additional studies will be required to address these otherwise incongruous results. Regardless, pregnenolone concentrations were still 10-fold higher in luteal than cotyledonary tissues even though progesterone concentrations were four orders of magnitude higher in luteal than cotyledonary tissues. This speaks to the remarkable steroid synthetic capacity of the corpus luteum of late gestation in cattle even if function is in decline (Mills & Morrissette 1970, Shemesh 1990).
The measurement and effective partitioning of 3βHSD into the microsomal (endoplasmic reticulum) and mitochondrial compartments (Thomas et al. 1988, 1989) has rarely been investigated in a physiological context, certainly not in bovine tissues. Enzyme activity and transcript data were consistent with one another, indicating that cotyledonary expression of 3βHSD was higher than caruncular expression. Within the cotyledonary tissues then, the data presented here suggest that both microsomal and mitochondrial intra-cellular compartments contribute significantly to progesterone synthesis by the bovine placenta. It is noteworthy also that the demonstration of 3βHSD activity in the present experiments is consistent with the enzyme being located on the cytoplasmic side of both microsomes and mitochondria. Activities were robust and apparently fueled effectively by NAD+ supplied in the buffer, which would be unable to cross intact membrane in charged form. The higher specific activity in the mitochondrial fraction was off-set by the larger size (based on recovered protein) of the microsomal compartment in the cotyledonary tissues. No differences were noted in 3βHSD activity between microsomes and mitochondria in placentas from SAL- and DEX-treated cows, so whether or not there is regulation of compartmental distribution of the enzyme is unclear. However, there were higher levels of 3βHSD activity in luteal microsomes than mitochondria, both of which were substantially higher than in cotyledonary tissue. There were also higher levels of activity in luteal mitochondria from DEX-treated than SAL-treated cows. This might reflect a membrane-stabilizing effect of the glucocorticoid (Madsen-Bouterse et al. 2006). Whether or not there is a mechanism controlling the partitioning of 3βHSD into microsomal or mitochondrial compartments of steroidogeneic tissues requires additional investigation and remains an intriguing possibility.
The activity of 5α-reductase in placental microsomes was slightly lower than that of 3βHSD, but of a similar order of magnitude. Based on these data, and correlations between luteal and systemic progesterone concentrations, it is reasonable to conclude that cotyledonary 3βHSD does not contribute significantly to circulating progesterone levels when the corpus luteum is functional. Since there was no detectable 20α-DHP in cotyledonary tissue, it seems equally unlikely that placental 5α-reductase contributes to circulating 20α-DHP. DEX had no effect on 20α-DHP either, but increased SRD5A2 expression in caruncular tissue which would certainly indicate that caruncular SRD5A2 expression has little influence on 20αDHP concentrations in systemic blood. In any case, 5α-reduced pregnane concentrations were universally low in placental tissues. Given the modest levels of 5α-reduced pregnanes in luteal tissue compared with progesterone itself, it is equally unlikely that this is the source of these metabolites either, even though this is the case in the elephant (Hodges et al. 1997). The rate of peripheral 5α-reduction of progesterone is certainly impressive in ovariectomized mares and geldings (Conley et al. 2018) and is likely of significance in cattle and women (Milewich et al. 1977, 1995). In aggregate, the results of this study provide little evidence to support the proposition that placental pregnene synthesis or metabolism has a major influence on maternal progesterone or 5α-reduced pregnane concentrations in cows with a functional corpus luteum at term. Similarly, it seems unlikely that reduced circulating pregnanes/enes at parturition involve changes in placental synthesis or metabolism.
In conclusion, the data presented here suggest it is more likely that that DEX initiates progesterone withdrawal and parturition in cattle in late gestation by inducing luteal regression than by effects on placental progestin synthesis or metabolism. This includes pregnenolone, the concentrations of which were unaffected by DEX in either placental or luteal tissues. High circulating concentrations of 20αDHP suggest that 5α-reduction is a major route of progesterone metabolism in the cow, and low placental and luteal concentrations suggest that metabolism likely occurs peripherally. DEX appears not to have a dramatic or rapid effect on 3βHSD enzyme activity in the placenta or in luteal tissue overall, even though transcript abundance for enzymes and co-factors supporting progesterone synthesis were decreased in luteal tissues of DEX-treated cows. Further studies are required to determine the mechanisms behind DEX-induced functional regression of the CL in cattle in late gestation.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/REP-18-0558.
Declaration of interest
Lawrence P Reynolds is on the editorial board of Reproduction. Lawrence P Reynolds was not involved in the review or editorial process for this paper, on which he is listed as an author. The other authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
The authors gratefully acknowledge the Academic Scholarship, ‘Steroids in regulation of parturition, fetal maturation, and placental expulsion,’ from the Society for Reproduction and Fertility (to L P R and A J C) and support from the North Dakota Agricultural Experiment Station.
Acknowledgements
The authors thank the Central Grasslands Research Extension Center, the Animal Nutrition and Physiology Center, and the Physiology Laboratory of the Department of Animal Sciences, NDSU, for their assistance with the animal husbandry, tissue collections and laboratory analyses; especially important were Bryan Neville, Mellissa Crosswhite, Sarah Underdahl, Nicolas Negrin, Matthew Crouse and Amelia Tanner. They also thank Lauren Hulsman Hanna for assistance with statistical analyses. They acknowledge the financial support of an Academic Scholarship, ‘Steroids in regulation of parturition, fetal maturation, and placental expulsion,’ from the Society for Reproduction and Fertility, UK, to L P R and A J C. Financial support of the North Dakota Agricultural Experiment Station is also acknowledged. The authors are equally grateful for the technical assistance of the staff at the Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, Davis, especially the efforts and support of Daniel McKemie, Teresa Bowers, Dr. Go Sugiarto and Sandy Yim, providing expertise, training and support for analysis of samples by liquid chromatography tandem mass spectrometry.
References
Adams WM & Wagner WC 1970 The role of corticoids in parturition. Biology of Reproduction 3 223–228. (https://doi.org/10.1093/biolreprod/3.2.223)
AVMA 2013 AVMA Guidelines for the Euthanasia of Animals. Schaumburg, IL, USA: American Veterinary Medical Association (AMVA).
Bowerman AM & Melampy RM 1962 Progesterone and delta-4-pregnen-20alpha-ol-3-one in bovine reproductive organs and body fluids. Experimental Biology and Medicine 109 45–48. (https://doi.org/10.3181/00379727-109-27099)
Brunton PJ, Russell JA & Hirst JJ 2014 Allopregnanolone in the brain: protecting pregnancy and birth outcomes. Progress in Neurobiology 113 106–136. (https://doi.org/10.1016/j.pneurobio.2013.08.005)
Challis JRG, Matthews SG, Gibb W & Lye SJ 2000 Endocrine and paracrine regulation of birth at term and preterm. Endocrine Reviews 21 514–550. (https://doi.org/10.1210/edrv.21.5.0407)
Comline RS, Hall LW, Lavelle RB, Nathanielsz PW & Silver M 1974 Parturition in the cow: endocrine changes in animals with chronically implanted catheters in the foetal and maternal circulations. Journal of Endocrinology 63 451–472. (https://doi.org/10.1677/joe.0.0630451)
Conley AJ & Ford SP 1987 Effect of prostaglandin F2 alpha-induced luteolysis on in vivo and in vitro progesterone production by individual placentomes of cows. Journal of Animal Science 65 500–507. (https://doi.org/10.2527/jas1987.652500x)
Conley AJ & Neto ACA 2008 The ontogeny of fetal adrenal steroidogenesis as a prerequisite for the initiation of parturition. Experimental and Clinical Endocrinology and Diabetes 116 385–392. (https://doi.org/10.1055/s-2008-1076713)
Conley AJ & Reynolds LP 2014 Steroidogenesis and the initiation of parturition. In Reproduction in Domestic Ruminants. Eds Juengel JL, Miyamoto A, Price C, Reynolds LP, Smith MF & Webb R. London: Society for Reproduction and Fertility pp 399–414.
Conley AJ, Head JR, Stirling DT & Mason JI 1992 Expression of steroidogenic enzymes in the bovine placenta and fetal adrenal glands throughout gestation. Endocrinology 130 2641–2650. (https://doi.org/10.1210/endo.130.5.1374010)
Conley AJ, Corbin CJ, Thomas JL, Gee NA, Lasley BL, Moeller BC, Stanley SD & Berger T 2012 Costs and consequences of cellular compartmentalization and substrate competition among human enzymes involved in androgen and estrogen synthesis. Biology of Reproduction 86 1–8. (https://doi.org/10.1095/biolreprod.111.094706)
Conley AJ, Scholtz EL, Legacki EL, Corbin CJ, Knych HK, Dujovne GD, Ball BA, Moeller BC & Stanley SD 2018 5alpha-dihydroprogesterone concentrations and synthesis in non-pregnant mares. Journal of Endocrinology 238 25–32. (https://doi.org/10.1530/JOE-18-0215)
Corbin CJ, Legacki EL, Ball BA, Scoggin KE, Stanley SD & Conley AJ 2016 Equine epididymal 5α-reductase expression and activity. Journal of Endocrinology 231 23–33. (https://doi.org/10.1530/JOE-16-0175)
Erb RE, Estergreen VL, Gomes JR, Plotka ED & Frost OL 1968 Progestin levels in corpora lutea and progesterone in ovarian venous and jugular vein blood plasma of pregnant cows. Journal of Dairy Science 51 401–410. (https://doi.org/10.3168/jds.S0022-0302(68)86998-X)
Estergreen VL, Frost OL, Gomes WR, Erb RE & Bullard JF 1967 Effect of ovariectomy on pregnancy maintenance and parturition in dairy cows. Journal of Dairy Science 50 1293–1295. (https://doi.org/10.3168/jds.S0022-0302(67)87615-X)
Hirayama H, Ushizawa K, Takahashi T, Sawai K, Moriyasu S, Kageyama S, Miura R, Matsui M, Fukuda S & Naito A et al. 2012 Differences in apoptotic status in the bovine placentome between spontaneous and induced parturition. Journal of Reproduction and Development 58 585–591. (https://doi.org/10.1262/jrd.2012-043)
Hodges JK, Heistermann M, Beard A & Van Aarde RJ 1997 Concentrations of progesterone and the 5 alpha-reduced progestins, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one, in luteal tissue and circulating blood and their relationship to luteal function in the African elephant, Loxodonta africana. Biology of Reproduction 56 640–646. (https://doi.org/10.1095/biolreprod56.3.640)
Hoffmann B & Schuler G 2002 The bovine placenta; a source and target of steroid hormones: observations during the second half of gestation. Domestic Animal Endocrinology 23 309–320. (https://doi.org/10.1016/S0739-7240(02)00166-2)
Hoffmann B, Wagner WC, Hixon JE & Bahr J 1979 Observations concerning the functional status of the corpus luteum and the placenta around parturition in the cow. Animal Reproduction Science 2 253–266. (https://doi.org/10.1016/0378-4320(79)90051-4)
Juengel JL, Garverick HA, Johnson AL, Youngquist RS & Smith MF 1993 Apoptosis during luteal regression in cattle. Endocrinology 132 249–254. (https://doi.org/10.1210/endo.132.1.8419126)
Larson JE, Lamb GC, Stevenson JS, Johnson SK, Day ML, Geary TW, Kesler DJ, Dejarnette JM, Schrick FN & Dicostanzo A et al. 2006 Synchronization of estrus in suckled beef cows for detected estrus and artificial insemination and timed artificial insemination using gonadotropin-releasing hormone, prostaglandin F2alpha, and progesterone. Journal of Animal Science 84 332–342. (https://doi.org/10.2527/2006.842332x)
Legacki EL, Scholtz EL, Ball BA, Stanley SD, Berger T & Conley AJ 2016 The dynamic steroid landscape of equine pregnancy mapped by mass spectrometry. Reproduction 151 421–430. (https://doi.org/10.1530/REP-15-0547)
Legacki EL, Ball BA, Corbin CJ, Loux SC, Scoggin KE, Stanley SD & Conley AJ 2017 Equine fetal adrenal, gonadal and placental steroidogenesis. Reproduction 154 445–454. (https://doi.org/10.1530/REP-17-0239)
Legacki EL, Corbin CJ, Ball BA, Scoggin KE, Stanley SD & Conley AJ 2018 Steroidogenic enzyme activities in the pre- and post-parturient equine placenta. Reproduction 155 51–59. (https://doi.org/10.1530/REP-17-0472)
Livak KJ & Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 402–408. (https://doi.org/10.1006/meth.2001.1262)
Madsen-Bouterse SA, Rosa GJ & Burton JL 2006 Glucocorticoid modulation of Bcl-2 family members A1 and Bak during delayed spontaneous apoptosis of bovine blood neutrophils. Endocrinology 147 3826–3834. (https://doi.org/10.1210/en.2006-0142)
Martins-Júnior HA, Pinaffi FL, Simas RC, Tarouco AK, Ferreira CR, Silva LA, Nogueira GP, Meirelles FV, Eberlin MN & Perecin F 2014 Plasma steroid dynamics in late- and near-term naturally and artificially conceived bovine pregnancies as elucidated by multihormone high-resolution LC-MS/MS. Endocrinology 155 5011–5023. (https://doi.org/10.1210/en.2013-2166)
Mcdonald LE, Mcnutt SH & Nichols RE 1953 On the essentiality of the bovine corpus luteum of pregnancy. American Journal of Veterinary Research 14 539–541.
Melampy RM, Hearn WR & Rakes JM 1959 Progesterone content of of bovine reproductive organs and blood during pregnancy. Journal of Animal Science 18 307–313. (https://doi.org/10.2527/jas1959.181307x)
Milewich L, Gomez-Sanchez C, Crowley G, Porter JC, Madden JD & Macdonald PC 1977 Progesterone and 5alpha-pregnane-3,20-dione in peripheral blood of normal young women: daily measurements throughout the menstrual cycle. Journal of Clinical Endocrinology and Metabolism 45 617–622. (https://doi.org/10.1210/jcem-45-4-617)
Milewich L, Mendonca BB, Arnhold I, Wallace AM, Donaldson MDC, Wilson JD & Russell DW 1995 Women with steroid 5alpha-reductase-2 deficiency have normal concentrations of plasma 5alpha-dihydroprogesterone during the luteal-phase. Journal of Clinical Endocrinology and Metabolism 80 3136–3139.
Mills RC & Morrissette MC 1970 Progesterone synthesis by perfused bovine ovaries of early and late pregnancy. Journal of Reproduction and Fertility 22 435–440. (https://doi.org/10.1530/jrf.0.0220435)
Moran FM, Ford JJ, Corbin CJ, Mapes SM, Njar VC, Brodie AM & Conley AJ 2002 Regulation of microsomal P450, redox partner proteins, and steroidogenesis in the developing testes of the neonatal pig. Endocrinology 143 3361–3369. (https://doi.org/10.1210/en.2002-220329)
Nguyen PT, Conley AJ, Soboleva TK & Lee RS 2012 Multilevel regulation of steroid synthesis and metabolism in the bovine placenta. Molecular Reproduction and Development 79 239–254. (https://doi.org/10.1002/mrd.22021)
Reynolds LP, Millaway DS, Kirsch JD, Infeld JE & Redmer DA 1990 Growth and in-vitro metabolism of placental tissues of cows from day 100 to day 250 of gestation. Journal of Reproduction and Fertility 89 213–222. (https://doi.org/10.1530/jrf.0.0890213)
Reynolds LP, Haring JS, Johnson ML, Ashley RL, Redmer DA, Borowicz PP & Grazul-Bilska AT 2015 Placental development during early pregnancy in sheep: estrogen and progesterone receptor messenger RNA expression in pregnancies derived from in vivo-produced and in vitro-produced embryos. Domestic Animal Endocrinology 53 60–69. (https://doi.org/10.1016/j.domaniend.2015.05.003)
Reynolds LP, Legacki EL, Corbin CJ, Caton JS, Vonnahme KA, Stanley S & Conley AJ 2018 Ovine placental steroid synthesis and metabolism in late gestation. Biology of Reproduction 99 662–670. (https://doi.org/10.1093/biolre/ioy089)
Scholtz EL, Krishnan S, Ball BA, Corbin CJ, Moeller BC, Stanley SD, Mcdowell KJ, Hughes AL, Mcdonnell DP & Conley AJ 2014 Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5alpha-dihydroprogesterone. PNAS 111 3365–3370. (https://doi.org/10.1073/pnas.1318163111)
Schuler G, Ozalp GR, Hoffmann B, Harada N, Browne P & Conley AJ 2006 Reciprocal expression of 17alpha-hydroxylase-C17,20-lyase and aromatase cytochrome P450 during bovine trophoblast differentiation: a two-cell system drives placental oestrogen synthesis. Reproduction 131 669–679. (https://doi.org/10.1530/rep.1.01033)
Schuler G, Greven H, Kowalewski MP, Doring B, Ozalp GR & Hoffmann B 2008 Placental steroids in cattle: hormones, placental growth factors or by-products of trophoblast giant cell differentiation? Experimental and Clinical Endocrinology and Diabetes 116 429–436. (https://doi.org/10.1055/s-2008-1042408)
Schuler G, Furbass R & Klisch K 2018 Placental contribution to the endocrinology of gestation and parturition. Animal Reproduction 15 822–842. (https://doi.org/10.21451/1984-3143-AR2018-0015)
Shemesh M 1990 Production and regulation of progesterone in bovine corpus luteum and placenta in mid and late gestation: a personal review. Reproduction, Fertility, and Development 2 129–135. (https://doi.org/10.1071/RD9900129)
Shenavai S, Preissing S, Hoffmann B, Dilly M, Pfarrer C, Ozalp GR, Caliskan C, Seyrek-Intas K & Schuler G 2012 Investigations into the mechanisms controlling parturition in cattle. Reproduction 144 279–292. (https://doi.org/10.1530/REP-11-0471)
Sherbet DP, Papari-Zareei M, Khan N, Sharma KK, Brandmaier A, Rambally S, Chattopadhyay A, Andersson S, Agarwal AK & Auchus RJ 2007 Cofactors, redox state, and directional preferences of hydroxysteroid dehydrogenases. Molecular and Cellular Endocrinology 265–266 83–88. (https://doi.org/10.1016/j.mce.2006.12.021)
Silver M 1990 Prenatal maturation, the timing of birth and how it may be regulated in domestic animals. Experimental Physiology 75 285–307. (https://doi.org/10.1113/expphysiol.1990.sp003405)
Stormshak F & Erb RE 1961 Progesteorne in bovine corpora lutea, ovaries and adrenals during pregnancy. Journal of Dairy Science 44 310–320. (https://doi.org/10.3168/jds.S0022-0302(61)89736-1)
Thomas JL, Berko EA, Faustino A, Myers RP & Strickler RC 1988 Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5→ 4-ene-isomerase: purification from microsomes, substrate kinetics, and inhibition by product steroids. Journal of Steroid Biochemistry 31 785–793. (https://doi.org/10.1016/0022-4731(88)90287-7)
Thomas JL, Myers RP & Strickler RC 1989 Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5→ 4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes. Journal of Steroid Biochemistry 33 209–217. (https://doi.org/10.1016/0022-4731(89)90296-3)
Thorburn GD & Challis JR 1979 Endocrine control of parturition. Physiological Reviews 59 863–918. (https://doi.org/10.1152/physrev.1979.59.4.863)
Thorburn GD, Challis JR & Currie WB 1977 Control of parturition in domestic animals. Biology of Reproduction 16 18–27. (https://doi.org/10.1095/biolreprod16.1.18)
Tsumagari S, Kamata J, Takagi K, Tanemura K, Yosai A & Takeishi M 1994 3 beta-hydroxysteroid dehydrogenase activity and gestagen concentrations in bovine cotyledons and caruncles during gestation and parturition. Journal of Reproduction and Fertility 102 35–39. (https://doi.org/10.1530/jrf.0.1020035)
Vonnahme KA, Zhu MJ, Borowicz PP, Geary TW, Hess BW, Reynolds LP, Caton JS, Means WJ & Ford SP 2007 Effect of early gestational undernutrition on angiogenic factor expression and vascularity in the bovine placentome. Journal of Animal Science 85 2464–2472. (https://doi.org/10.2527/jas.2006-805)
Waite AL, Holtan DW & Stormshak F 2005 Changes in bovine luteal progesterone metabolism in response to exogenous prostaglandin F(2alpha). Domestic Animal Endocrinology 28 162–171. (https://doi.org/10.1016/j.domaniend.2004.08.001)
