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. Author manuscript; available in PMC: 2025 Jul 14.
Published in final edited form as: Environ Res. 2024 Nov 5;264(Pt 1):120303. doi: 10.1016/j.envres.2024.120303

In-utero exposure to real-life environmental chemicals disrupts gene expression within the hypothalamo-pituitary-gonadal axis of prepubertal and adult rams

Mohammad Ghasemzadeh Hasankolaei a,*, Neil P Evans a, Chris S Elcombe a,1, Richard G Lea b, Kevin D Sinclair b, Vasantha Padmanabhan c, Michelle Bellingham a
PMCID: PMC12259014  NIHMSID: NIHMS2089293  PMID: 39510237

Abstract

Environmental chemicals (ECs) have been associated with a broad range of disorders and diseases. Daily exposure to various ECs in the environment, or real-life exposure, has raised significant public health concerns. Utilizing the biosolids-treated pasture (BTP) sheep model, this study demonstrates that in-utero exposure to a real-life EC mixture disrupts hypothalamo-pituitary-gonadal (HPG) axis gene expression and reproductive traits in prepubertal (8-week-old, 8w) and adult (11-month-old) male sheep. Ewes were maintained on either BTP or pastures fertilized with inorganic fertilizer [control (C)] from approximately one month prior to insemination until around parturition. Thereafter, all animals were kept under control conditions. Effects on reproductive parameters including testosterone concentrations and the expression of key genes in the HPG axis were evaluated in eight-week-old and adult male offspring from both C and biosolids-exposed (B) groups. Results showed that, at 8w, relative to C (n=11), B males (n=11) had lower body weight, and altered testicular expression of HSD3B1, LHR and HSD17B3, BMP4, ABP, P27kip and CELF1. Principal component analysis (PCA) identified two 8w B subgroups, based on hypothalamic expression of GnRH, ESR1, and AR, and pituitary expression of KISSR. The two subgroups also exhibited different serum testosterone concentrations. The largest biosolids effects were observed in the hypothalamus of adult rams with NKB, ESR1, KISS1, AR, DLK1 and GNRH1 mRNA expression differing between B (n=10) and C (n=11) rams. Testicular steroidogenic enzymes CYP11A1 and HSD3B1 mRNA expression also differed between exposure groups. PCA identified two adult B subgroups, with BS1 (n=6) displaying hypothalamic effects and BS2 (n=4) both hypothalamic and testicular effects. The subgroups also differed in circulating testosterone concentrations. These findings demonstrate that exposure to a real-life EC mixture may predispose some males to infertility, by disrupting key functional HPG markers before puberty with consequent downstream effects on steroid hormones and spermatogenesis.

Keywords: real-life exposure, male offspring, environmental chemicals, prepubertal, adult, Hypothalamic-Pituitary-Gonadal Axis

1. Introduction

Environmental chemicals (ECs) can disrupt the body’s hormonal and homeostasis systems, leading to alterations in physiological state (Kumar et al., 2020). The evidence for reproductive dysregulation as a consequence of exposure to ECs is escalating, with critical reviews published by the “Endocrine Society” in 2009 (Diamanti-Kandarakis et al., 2009) and 2015 (Gore et al., 2015). EC exposure is recognised as a contributory factor for the development of obesity, diabetes mellitus, cardiovascular disease, thyroid disruption, hormone sensitive cancers and male and female reproductive health problems, including infertility (Gore et al., 2015; Rodgers et al., 2018; Salazar et al., 2021; Weng et al., 2023).

With regard to male reproductive health, exposure to plasticisers such as bisphenol A (BPA) and phthalates, as well as phenols, polychlorinated biphenyls (PCBs), pesticides, per- and polyfluoroalkyl substances (PFAS) and polybrominated diphenyl ethers (PBDEs) have documented adverse effects on reproductive function (Diamanti-Kandarakis et al., 2009; Gore et al., 2015; Wang et al., 2016). Increased post-implantation embryo loss, decreased gamete production, reduced FSH and LH, disrupted Leydig cell function, reduced sperm motility and testosterone secretion, increased sperm DNA fragmentation, and induction of prostatic disease have been reported as consequences of exposure to the mentioned ECs in males (Anway et al., 2005; Doshi et al., 2012; Foster, 2006; Foster et al., 2001; Foster et al., 2011; Mai et al., 2020; Manikkam et al., 2012; Saillenfait et al., 2009; Sanabria et al., 2016; Sumner et al., 2019; Zhao et al., 2014). While some of these effects may be mediated by direct EC action at the level of the gonad or accessory sex organs, studies which have reported effects of dioxin exposure on puberty (Manikkam et al., 2012), pituitary LH synthesis (Mutoh et al., 2006) and GNRH1 gene expression (Takeda et al., 2014) indicate central EC effects. Furthermore, in vivo exposure to PCBs, BPA, PFOS and dioxins result in reproductive disorders and low sperm quality, effects on hypothalamic development and gene expression, and/or effects across the entire hypothalamic-pituitary-gonadal (HPG) axis (Bell, 2014; Egalini et al., 2022; Wisniewski et al., 2015; Yeung et al., 2011).

Most of the above studies have evaluated the effects of individual ECs on male reproductive health. Results are now becoming available, however, that describe adverse effects on the HPG axis after in vivo exposure to EC mixtures. For example, in utero exposure of rats to a mixture of PCBs (Aroclor 1221 - A1221) results in altered hypothalamic gene expression in male offspring (Dickerson et al., 2011; Walker et al., 2013). When considering potential risks of EC exposure to human health, such EC mixture studies are important (Taboureau et al., 2020), as the human exposome consists of a complex mixture of thousands of ECs all at low individual levels (Elcombe et al., 2022a; Zhao et al., 2021). The biosolids-treated pasture sheep model was developed to gain an understanding of the consequences of in utero exposure to a real-life mixture of ECs. Biosolids (the solid and semi-solid residues from wastewater treatment) are used extensively as an agricultural/horticultural fertilizer and in land remediation throughout Europe and the USA. According to reports, 726 ECs have been identified in biosolids including PFAS, organotins, triclosan, PCBs, dioxins, PBDEs and dibenzofurans, all typically present at very low concentrations (Fijalkowski et al., 2017; Richman et al., 2022). This broad range of low-concentration ECs provides a representation of the human exposome. Previous studies with this model have described how grazing of pregnant animals on biosolids treated pasture (BTP) (according to current regulations), and therefore in utero exposure to real-life EC mixtures, has an impact on bone density (Lind et al., 2009; Lind et al., 2010), behaviour (Erhard and Rhind, 2004), metabolic systems (Filis et al., 2019; Ghasemzadeh-Hasankolaei et al., 2023; Hombach-Klonisch et al., 2013), and the reproductive system both at the hypothalamo-pituitary (Bellingham et al., 2009; Bellingham et al., 2016) and gonadal levels. With respect to latter, phenotypic and gene expression changes have been reported in the ovaries (Lea et al., 2016) and testes (Elcombe et al., 2022b; Elcombe et al., 2021; Elcombe et al., 2023; Lea et al., 2022; Paul et al., 2005)) of lambs derived from BTP exposed mothers. The aim of the current study was to gain an understanding of the on the expression profile of key regulatory genes in the HPG axis of pre- and post-pubertal rams born to mothers who received periconceptional and gestational exposure to a real-life EC mixture derived from BTPs.

2. Material and methods

2.1. Study design

Adult EasyCare ewes (n= 320) were divided into two groups, from approximately one month before pregnancy until two weeks before parturition. One group was kept on control pasture (C), fertilized with standard inorganic fertilizers at conventional rates. The other group was maintained on biosolids (B)-treated pasture, which received 4 tonnes/ha of biosolids twice per annum. The two fertilization methods provided equivalent levels of nitrogen to pastures (225 kg N/ha per annum). To minimize any impact of paternal genotype on molecular and phenotypic traits, four sires were utilized in the derivation of offspring across both treatment groups. During data analysis, paternal genotypes was simultaneously assessed with biosolids exposure group. There were no significant differences in pregnancy rate, lamb survival or sex ratio between the C and B groups. 11 first generation (F1) ram lambs from each group (C and B) were euthanized at 8 weeks of age (8w, prepubertal stage), by barbiturate overdose (140 mg/kg Dolethal, Vetroquinol, UK). Another cohort of F1 rams (C n=11, B n=10) were euthanized, at 11 months of age (adult), as described above. These animals had been the subject of a series of studies characterizing the effects of real-life EC exposure (Elcombe et al., 2022b; Elcombe et al., 2021; Elcombe et al., 2023). In the current study, a total of 52 different variables, including the expression level of various genes’ mRNA and proteins, as well as some phenotypical parameters, were evaluated.

2.2. Blood/tissue sampling and preparation

Animals were weighed approximately one week prior to euthanasia, and blood samples collected by jugular venipuncture (BD Vacutainer Plus, BD, USA). Plasma was harvested after centrifugation (3000×g, 15 min, 4 °C) and stored at −20 °C for future testosterone measurement.

Immediately following euthanasia, the hypothalamus, pituitary gland and testes were dissected, and pituitary gland and testes weight recorded. Half of the hypothalamus and pituitary were snap frozen in liquid nitrogen and stored at −80 °C, while the other half was fixed in 10% neutral buffered formalin (NBF, Thermo Scientific – 16499713). Central coronal sections from each testis were also fixed or frozen similarly. For molecular analysis, specific hypothalamic areas were isolated as described previously (Bellingham et al., 2016). Briefly, using visible anatomical landmarks (e.g., the anterior commissure, the mammillary body) the hemi-hypothalamic block was cut into 2mm coronal slices, and 2mm diameter punches of specific hypothalamic areas collected into 500μl TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). Punches from the mPOA (medial preoptic area), AHA (anterior hypothalamic area), rostral ARC (arcuate nucleus) and caudal ARC were used, as these regions play pivotal roles in regulating reproductive function (Herman et al., 2017; Whitelaw et al., 2012). A coronal section from the central face of the each frozen hemi pituitary and a small section representative of the centre of the testes was taken from the frozen sample and placed into 500μl TRIzol® reagent. Thereafter, samples were homogenised using a FastPrep-24 5G homogenizer (MP Biomedicals, Germany) at 4 m/s for 45 seconds.

2.3. RNA extraction, cDNA synthesis and real time RT-PCR

After samples were homogenized, mRNA was extracted using Qiagen RNeasy® RNA extraction Mini kit (Qiagen, Hilden, Germany) spin columns according to company instructions. mRNA concentration and purity were assessed using a ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and 500 ng mRNA reverse transcribed using QuantiTect Reverse Transcription Kits (Qiagen, Hilden, Germany). Expression of the genes of interest was assessed using 12.5 ng cDNA by quantitative real-time PCR using brilliant II SYBR Master Mix (Agilent technologies, USA) on a Stratagene 3000 machine. The resultant data were analysed using REST© software (Pfaffl et al., 2002). The specific markers investigated for each tissue were as follows; mPOA and AHA of the hypothalamus - GNRH1; cranial and caudal ARC of the hypothalamus – NKB (TAC3), KISS1, DLK1, and AR; anterior pituitary gland- LHB, GNRHR, GNRH1, KISSR, KISS1, AR and GH; testes – ABP (androgen binding protein or sex hormone binding globulin, SHBG), SCF, P27kip1 (CDKN1B), GDNF, LHR (LHCGR), HSD3B1, DAZL, STAR, CYP17A1, CYP11A1, HSD17B3, mLST8, BMP4, mTOR, AR, VASA, MOV10L1, FKBP6, SOX9, CELF1, CEP57 and CIB1. These markers were selected for their critical roles in regulating male reproduction and spermatogenesis at the hypothalamus (Sobrino et al., 2022; Xie et al., 2022), pituitary (Gahete et al., 2016; Hull and Harvey, 2014), and testis (Cibois et al., 2012; Du et al., 2021; Gholamitabar Tabari et al., 2018; Matusik et al., 2021; Yuan et al., 2006). Since the impacts of exposure to EC mixtures on testicular development and function (Elcombe et al., 2022b; Elcombe et al., 2021; Elcombe et al., 2023; Evans et al., 2023) can involve epigenetic alterations in germ cells (King and Skinner, 2020), to gain an understanding of the mechanistic basis, the expression of the epigenetic regulatory markers, DNMT1, DNMT3a and DNMT3b, were evaluated in the testis samples. Primer sequences are shown in Supplementary Table 1. Relative expression (log2) for each transcript was calculated following normalization to the endogenous reference gene, ACTB.

2.4. Histological assessment of the pituitary

Pituitary glands of 8w and adult males underwent histological assessments to detect LH immunopositive cells. Three 5μm midline sections were cut from the NBF-fixed, paraffin-embedded pituitary gland samples using a Microtome (Leica Biosystems, model RM2125RT). Sections were dewaxed, subject to antigen retrieval (autoclaved in citrate buffer (10mM, pH 6) for 21 minutes) and washed in TBS. Then, peroxidase (3% hydrogen peroxide/10 min), avidin, and biotin blocking solutions (Vector Labs, 15 minutes each with TBS washes in between) were applied. Non-specific binding was blocked using 20% goat serum in TBS for 30 minutes, followed by incubation with goat anti-rabbit LHβ primary antibody (A. Parlow Los Angeles Medical Centre, CA, USA, NHPP, 1:10000 dilution) at 4°C overnight. Next, the slides were washed in TBST (3 × 5 min), incubated with the biotinylated goat anti-rabbit 2° antibody for 1 hour at room temperature, washed and incubated with Vectastain ABC solution (Vector Labs) for 30 min. LHβ positive cells were visualised using DAB. For each animal the mean number of LHβ positive cells was counted (manually) in three separate images taken from the rostral, mid and caudal regions for the three pituitary gland sections using a Leica DM4000B microscope with a Leica DC480 camera at ×40 magnification. The mean number of cells was calculated for each animal and exposure group (C or B).

To count the number of cells in the pituitary tissue of adult rams, the sections were stained with haematoxylin and eosin (H&E). Three images were captured from the top, middle, and bottom of one section per animal at 20× magnification (Supplementary Figure 1). The number of cells was quantified using ImageJ software and the average cell count from the three different areas within each section used as the representative number of cells for that animal.

2.5. Testosterone measurement

The circulating testosterone concentrations were determined using a colorimetric Testosterone ELISA (R&D Systems – KGE010) according to manufacturer’s instructions and previously published report (Evans et al., 2023). The assay sensitivity averaged 0.027 ng/ml, with inter- and intra-assay coefficients of variation averaged 7.4% and 4.8%, respectively.

2.6. Statistical analysis

Pituitary gland and testes weights were normalized to body weight (BW) prior to analysis. Paired testes volume (PTV) was calculated (for adult animals only) using the formula suggested by Godfrey and colleagues (Godfrey et al., 1998): PTV: 0.0396 × testis length × Scrotal circumference2 and normalised relative to BW.

All datasets were analysed using the MetaboAnalyst (MetaboAnalyst 5.0 http://www.metaboanalyst.ca) online platform. Student’s t-test was applied to compare the expression of the evaluated factors/genes between C and B animals, within each age, and p<0.05 was considered as statistically significant. Principal Component Analysis (PCA) and clustering of the samples, Orthogonal partial least squares discriminant analysis (OPLS-DA), spare (s) PLS-DA, plotting of heatmaps box plots and correlation assay were performed using MetaboAnalyst 5.0 web-tool. The subgroups within the 8w and adult B animals (detected by PCA) were compared with a one-way ANOVA and Tukey-HSD test using R (version 4.2.3) within R Studio (version 2023.03.0+386). The same software was employed to conduct a two-way ANOVA, followed by a Tukey-HSD post-hoc test with sire and biosolids exposure as variables on the factors/markers with differential expression in 8w or adult B group compared to its corresponding C group to examine the effects of sire genotype and biosolids exposure. Where relevant, KEGG pathway and Gene Ontology (GO) functional analysis was conducted using the DAVID online databases. The gene network and interactions were verified utilizing the STRING database (https://string-db.org/). All data are presented as means ± SEM unless indicated otherwise.

3. Results

3.1. Body weight (BW)

In the 8w lambs, BW was lower (P< 0.03) in B compared to C animals (Figure 1). For the 11-month-old rams, we reported previously a trend (p=0.08) indicating that body weight was lower in B rams compared to C adult rams (Ghasemzadeh-Hasankolaei et al., 2023).

Figure 1.

Figure 1.

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Lower mean (orange dot represents the mean, and the horizontal line represents the median of the data for each group) body weight was observed in 8w ram lambs from the biosolids group (B, n=11) compared to control group (C, n=11). Upper and lower whiskers represent maximum and minimum values.

3.2. Testicular weight and paired testes volume (PTV)

Testes weight (adjusted for body weight) did not differ between C and B rams in either the 8w (C, 1.76 ± 0.46 g/KgBW; B, 1.63 ± 0.66 g/KgBW), or adult (C,4.79 ± 0.49: B,5.25 ± 0.94 g/kg) animals. PTV also did not differ between B and C adult rams (C, 17.66 ± 4.5 cm3/kgBW: B, 16.29 ± 4 cm3/kgBW).

3.3. Pituitary weight and cell number

Mean pituitary gland weight (corrected for body weight) in the 8w B rams (20 ± 0.9 mg/KgBW) was similar to that in the C group animals (17 ± 0.7 mg/KgBW). However, in adults, mean pituitary gland weight was greater (p<0.05) in B (15 ± 0.9 mg/KgBW) than in C rams (13 ± 0.4 mg/KgBW) (Figure 2A, B). Although there wasn’t any statistically significant difference in pituitary cell density between 8w C and B ram lambs (Figure 2C), adult B rams showed lower cell density (p = 0.05) in their pituitary glands compared to controls, suggesting the presence of larger or hypertrophic cells (Figure 2D).

Figure 2.

Figure 2.

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Box plot of pituitary weight (relative to body weight) showing the mean (orange dot), median (the horizontal line) and the min and max values (whiskers) for A: 8w B (n=11) compared to C ram lambs (n=11). B: Adult B (n=10) compared to C rams (n=11). Bar graphs showing the mean cell count ± SE per evaluated area in the pituitary glands of C and B animals in the 8w (C) and adult (D) groups.

3.4. Tissue-specific gene expression pattern

3.4.1. Hypothalamus

Real time RT-PCR analysis of GNRH1 and ESR1 expression in the mPOA and AHA and NKB, KISS1, DLK1, AR and ESR1 in the rostral and caudal ARC indicated no differences between 8w C and B rams (Supplementary Figure 2A). However, in the adults, B rams exhibited greater (p<0.05) expression of GNRH1 in the mPOA, and lower (P<0.05) expression of NKB and KISS1 in the rostral ARC. Trends were also noted for the expression of AR (p=0.08), and DLK1 (p=0.1) to be lower in B compared to C rams in the rostral ARC. Additionally, DLK1 was lower (p=0.1) in the caudal ARC of B vs C adult rams (Figure 3). We have previously reported that the expression of ESR1 in the rostral ARC of this cohort of rams was significantly lower in B compared to C rams (Ghasemzadeh-Hasankolaei et al., 2023). No differences were noted in the expression of the markers tested in either the caudal ARC or the AHA, as a result of B exposure (Supplementary Figure 2B).

Figure 3.

Figure 3.

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Box plots showing the mean (orange dot), median (the horizontal line) and the min and max levels (whiskers) of relative expression levels of the markers that were significantly different between the hypothalamus of adult B and C rams. Hypothalamic regions are shown in parentheses. Ro. ARC: rostral ARC, Ca. ARC: Caudal ARC.

3.4.2. Pituitary

The expression levels of ESR1, LHB, GnRHR, GNRH1, KISSR, KISS1, AR and GH was not different between C and B rams in either the 8w or adult animals (Supplementary Figure 3A, B).

3.4.3. Testis gene expression

In 8w old animals, the expression of CELF1 (germ cell marker) was higher (p<0.05) in B relative to C animals. The expression of the T secretion pathway markers, HSD3B1, HSD17B3 and LHR, was higher (p<0.05), and ABP lower (P<0.05) in B compared to C animals. BMP4 expression was also higher (p<0.05) in B compared to C animals and a trend was noted for P27kip1 (p=0.07) and STAR (P=0.1) expression to be higher in B compared to C 8w lambs. B lambs also had higher (p<0.05) levels of expression of DNMT3a, in their testes relative to C lambs (Figure 4A). The remaining evaluated markers exhibited similar expression levels in B compared to C lambs (Supplementary Figure 4A). In adult testes, two testosterone secretion related markers, CYP11A1 (p<0.05) and HSD3B1 (p=0.06), were expressed at higher levels in B compared to C animals (Figure 4B), with no differences observed with the other markers tested (Supplementary Figure 4B).

Figure 4.

Figure 4.

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A: 8w in-utero biosolids chemical mixture exposed ram lambs (n=11) exhibited distinct expression patterns of various markers in the testis compared to C lambs (n=11). B: Adult B rams (n=10) displayed elevated mRNA levels of testicular CYP11A1 and HSD3b1 compared to C rams (n=11). Orange dot represents the mean, and the horizontal line represents the median of the data for each group. Upper and lower whiskers represent maximum and minimum values.

3.5. LHB cell populations in the pituitary

The mean number of LHβ immunopositively stained cellsdid not differ between the B and C groups in either the 8w or adult animals (Supplementary Figure 5A, B).

3.6. Blood testosterone concentrations

Mean plasma testosterone concentrations were not significantly different between the B and C, 8w and adult animals (Supplementary Figure 6A, B).

3.7. Principal component analysis (PCA)

3.7.1. 8w group

A heatmap displaying the differently expressed factors/markers (out of the 52 evaluated markers/factors) with p<0.1 is presented in Figure 5A. The OPLS-DA analysis revealed that the C and B datasets were placed in separate groups (Supplementary Figure 7A). PCA analysis revealed that three of the B lambs (8wBS1) were not situated in the same cluster as the C group and the rest of the B lambs (8wBS2, n=8) that grouped together (Figure 5B). Analysis of the 8wBS1 subgroup data revealed that the animals displayed altered gene expression within the hypothalamus and pituitary compared to both the C and 8wBS2 animals. 8wBS1 rams also displayed differences in the expression of the testicular markers BMP4, FKBP6, ABP, P27kip1 (all p<0.05), HSD3B1 (p=0.08), LHR (p=0.09), CELF1 (p=0.1) and CIB1 (p=0.1) when compared to the C group. Finally, the 8wBS1 subgroup exhibited lower levels of GNRH (p<0.05) and ESR1 (p=0.09) expression in the hypothalamic mPOA. The second subgroup, 8wBS2, displayed changes in the expression of BMP4 (p<0.05), HSD17B3 (p=0.06), HSD3B1 (p=0.1), and DNMT3a (p=0.1) in the testis, as well as KISSR (p=0.1) in the pituitary, relative to the C group. When the two 8wB subgroups were compared to each other, 8wBS1 rams possessed lower (p<0.05) transcript levels of GNRH in the hypothalamic mPOA compared to the 8wBS2 group. Conversely, 8WBS2 rams exhibited higher mRNA levels of KISSR (p<0.05) in the pituitary and AR in the rostral ARC (p=0.08) compared to 8wBS1 ram lambs (Table 1). The sPLS-DA analysis also separated the two B subgroups and C animals into almost separate groups. The sPLS-DA plot along with the heatmap of the values of all evaluated markers/factors in 8wBS1, 8wBS2 and C groups are shown in Supplementary Figure 7B, C.

Figure 5.

Figure 5.

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A: The heatmap illustrates the markers/factors that displayed distinct expression patterns in 8w B compared to C ram lambs. B: PCA analysis revealed that 3 members of B group (8wBS1) were positioned outside the cluster formed by C lambs and the rest of group B (8wBS2, n=8) ram lambs.

Table 1.

Various markers or factors exhibited differential expression across the study groups at 8 weeks of age.

Groups Altered markers/factors Tissue Change direction P value
8w C – 8w B (t-test) HSD3B1
BMP4
DNMT3a
LHR
HSD17B3
ABP
CELF1
P27kip1 		Testis Up
Up
Up
Up
Up
Down
Up
Up		 0.01
0.02
0.02
0.02
0.03
0.03
0.04
0.07
BW - Down 0.03
8wBS1 relative to 8w C
(One-way ANOVA)		 BMP4
FKBP6
ABP
P27kip1
HSD3B1
LHR
CELF1
CIB1		 Testis Up
Up
Down
Up
Up
Up
Up
Down		 0.01
0.03
0.03
0.05
0.08
0.09
0.1
0.1
GNRH
ESR1		 Hypothalamus mPOA Down
Down		 0.03
0.09
8wBS2 relative to 8w C (One-way ANOVA) BMP4
HSD17B3
HSD3B1
DNMT3a		 Testis Up
Up
Up
Up		 0.02
0.06
0.1
0.1
KISSR Pituitary Up 0.1
8wBS1 relative to 8wBS2 (One-way ANOVA) FKBP6

CIB1		 Testis 59% higher

9% lower		 0.03
0.1
AR Hypothalamus
Rostral ARC		 7.7% lower 0.08
GNRH Hypothalamus mPOA 24.3% lower 0.02
KISSR Pituitary 47.3% lower 0.02
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3.7.2. Adults group

Figure 6A depicts a heatmap illustrating the factors/markers (selected from a pool of 52 evaluated markers/factors) that exhibited varying levels/states with a significance threshold of p<0.1. The OPLS-DA analysis demonstrated that B and C rams can be classified into distinct groups (Supplementary Figure 8A). Again, PCA analysis revealed two subgroups within the B rams. AdultBS1 animals (n=4), grouped with C animals and AdultBS2 (n=6) which were distinct from the C group, Figure 6B. These subgroups displayed significant differences in the expression/state of certain evaluated markers/factors, particularly at the hypothalamic level, in comparison to both the C group and each other. AdultBS1 showed lower levels of expression of key reproduction markers, NKB (p<0.001), ESR1 (p<0.0001) and KISS1 (p<0.05) in the rostral ARC and ESR1 (p<0.05) in the mPOA of the hypothalamus compared to the C group. In the AdultBS2 animals the only hypothalamic marker affected was rostral ACR NKB (p<0.01) which was significantly lower than the controls, the remaining affected markers being testicular, CYP11A1 (lower, p<0.01), CYP17A1 (higher, p=0.09) and mLST8 (higher, p=0.09). Comparison of the two adult B subgroups revealed that AdultBS1 rams had significantly lower ESR1 transcript level in the rostral ARC and mPOA of the hypothalamus (p=0.001 and 0.01 respectively) compared to AdultBS2 rams. On the other hand, AdultBS2 subgroup showed higher expression levels of testicular steroidogenesis markers, CYP11A1 (p=0.1), CYP17A1 (p=0.03) and HSD3B1 (p=0.05), and lower testosterone level (p=0.05) relative to AdultBS1 group, Table 2. The results of the sPLS-DA analysis of data from C group and B subgroups and the heatmap of the values of various evaluated markers/factors can be seen in Supplementary Figure 8B, C).

Figure 6.

Figure 6.

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A: The heatmap of the markers/factors which expressed differently in adult B compared to C lambs. C: PCA analysis indicated that 4 members of the B group (AdultBS1) were located outside the cluster formed by C rams and the remaining members of the B group (AdultBS2, n=6) rams.

Table 2.

Markers/factors that were expressed differently across the study groups at 11 months of age.

Groups Altered markers/factors Tissue Change direction P value
Adult C – Adult B
(t-test)		 NKB
ESR1**
KISS1
AR
DLK1		 Hypothalamus Rostral ARC Down
Down
Down
Down
Down		 0.0001
0.01
0.01
0.08
0.1
CYP11A1
HSD3B1		 Testis Up
Up		 0.02
0.03
GNRH1 Hypothalamus mPOA Up 0.03
BW - Down 0.08
Pituitary weight Pituitary Up 0.04
DLK1 Hypothalamus
Caudal ARC		 Down 0.1
AdultBS1 relative to Adult C (One-way ANOVA) NKB
ESR1
KISS1		 Hypothalamus
Rostral ARC		 Down
Down
Down		 0.0005
0.00005
0.02
ESR1 Hypothalamus mPOA Up 0.03
AdultBS2 relative to Adult C (One-way ANOVA) NKB Hypothalamus
Rostral ARC		 Down 0.008
mLST8
CYP11A1
CYP17A1		 Testis Up
Down
Up		 0.09
0.008
0.09
AdultBS1 relative to AdultBS2 (One-way ANOVA) ESR1 Hypothalamus
Rostral ARC		 63% lower 0.001
CYP11A1
CYP17A1
HSD17B3		 Testis 4.8% lower
3.6% lower
3.4% lower		 0.1
0.03
0.05
Testosterone Blood 186% higher 0.05
ESR1 Hypothalamus
mPOA		 55% lower 0.01
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**

from our previous report (Ghasemzadeh-Hasankolaei et al., 2023).

3.8. Gene network construction and identification of pathways and GO related to the differentially expressed markers

Our results showed that, at 8w the B and C rams showed distinct differences in the expression of eight testicular markers (Supplementary Figure 9A). Network analysis (STRING database; Supplementary Figure 9B) indicated associations between changes in BMP4, HSD3B1, ABP (SHBG), and HSD17B3 with LHR (LHCGR) as a central hub. KEGG pathway analysis indicated that, except for CELF1 (the germ cell marker), the remaining differentially expressed testicular markers were closely linked to three pivotal pathways: ‘Ovarian steroidogenesis,’ ‘Steroid hormone biosynthesis,’ and ‘Cushing syndrome’. No significant findings emerged from the GO analysis.

In the adult rams, most of the markers affected by B exposure were derived from the hypothalamic rostral ARC (Supplementary Figure 9A). Network analysis (Supplementary Figure 9C) unveiled a network involving the five differently expressed markers found in the rostral ARC of B rams compared to C rams. Upon conducting pathway analysis, three enriched pathways were identified ‘Neuroactive ligand-receptor interaction’, ‘GnRH secretion’ and ‘Chemical carcinogenesis’. Remarkably, all the GO terms associated with “Biological Process,” were attributed to AR and ESR1 genes (Supplementary Table 2). Furthermore, CYP11A1 and HSD3B1, which exhibited distinct differences in expression in the testes of B compared to C rams, were found to significantly contribute to five crucial pathways: “Ovarian steroidogenesis”, “Cortisol synthesis and secretion”, “Steroid hormone biosynthesis”, “Aldosterone synthesis and secretion”, and “Cushing syndrome”. No GO term emerged following further analysis of the two testicular markers. It is worthy of note that HSD3B1 expression was affected in both 8w and adult B rams.

3.9. Age-related comparisons: 8w C vs. adult C rams and 8w B vs. adult B rams

When the data from the 8w and adult rams were compared (Supplementary Figure 10), with a p=0.1 threshold, 32 markers/factors (testicular, hypothalamic, and pituitary genes, blood testosterone, pituitary LHB protein, and pituitary and testis size) differed (either up- or downregulated) with age, in both the B and C animals. Age related changes were seen in the expression of a further 6 genes/location (GNRH1 in the mPOA and AHA, LHB, and GNRH1 in the pituitary, GDNF and STAR in the testis) in C and 4 genes (NKB, AR in the rostral ARC, ABP, and HSD17B3 in the testis) in the B animals.

3.10. Sire genotype vs biosolids exposure effects on gene expression

Among the identified differentially expressed markers in the testis of 8w rams, the expression of three markers was affected by paternal genotypes. ABP (exposure effect p=0.007 and sire effect =0.01), HSD3B1 (exposure effect p=0.02 and sire effect p=0.04), and DNMT3a (exposure effect p=0.04 and sire effect p=0.05). In the adult animals, different plasma testosterone between AdultBS1 and AdultBS2 was the only factor influenced by sire (exposure effect p=0.02 and sire effect p=0.02).

4. Discussion

The results of this study, which investigated the effects of exposure to a real-life EC mixture on the HPG axis in rams, demonstrate that although study animals were only exposed to ECs during foetal life, they exhibited phenotypic changes and distinct disruptions of gene expression both prior to puberty and as adult animals. Despite the body weights of the whole F1 population being comparable at birth (Evans et al., 2023), the mean body weights of the EC exposed rams included in this study were significantly lower than the controls both prior to puberty and as adults (11-months-old). The other effects of EC exposure noted in the prepubertal rams in this study related to testicular gene expression, which included disruption of markers associated with Leydig, Sertoli, and germ cells. The real-life EC mixture-induced changes in adults differed from prepubertal 8W rams in that, in addition to changes in two genes at the level of the testes (steroidogenic enzymes CYP17A1, HSD3B1), the effects were predominantly at the hypothalamo-pituitary level including an increase in the size of the pituitary gland, its component cells and hypothalamic expression of key regulatory genes.

The reduction in body weight in the EC mixture exposed rams observed between 8 and 44 weeks of age, matches previous findings in another cohort of the B animals that were followed to 54 weeks but showed catch-up growth after 44 weeks of age (Evans et al., 2023). EC-mixture exposure did not affect mean testes weight in either the prepubertal or adult rams or paired testes volume in the adult animals and agrees with results from adults published by Bellingham et al (Bellingham et al., 2012). While no effects of EC exposure were seen on testicular size in the ~10 months old rams or the mean testosterone concentrations between the C and B animals, the adult B rams had heavier pituitary glands compared to the controls, which was at least in part due to hypertrophy of pituitary cells. Pituitary enlargement has previously been reported in female rats exposed to tributyltin chloride (Merlo et al., 2016) but this is the first report of this effect in adult male sheep developmentally exposure to a real-life EC mixture. Previous ovine studies have reported pituitary hypertrophy in adult male and female sheep as a result of prenatal androgen exposure acting through the oestrogen receptor (Robinson et al., 2012). Biosolids contain a vast array of ECs, including oestrogenic compounds (Chawla et al., 2014), and so this result could suggest a dominant oestrogenic effect of biosolids exposure in our model at the level of the pituitary. Unlike in the in-utero androgen exposed animals (Robinson et al., 2012), however, no differences were observed in the EC exposed animals in the current study, in the number of LHβ immunopositive pituitary cells. The effect of a similar number of larger gonadotrophs on circulating LH concentrations has yet to be determined. We recently reported that the plasma androgen levels in the biosolids-exposed mothers of the rams in this study were elevated (Thangaraj et al., 2023). This is of relevance as the increase in androgens could disrupt development of the neuronal network responsible for regulating the reproductive axis in their offspring (Holland et al., 2019), and potentially explain the larger pituitary size in the B compared to C rams (Robinson et al., 2012).

Relative to gene transcriptional changes, real-life EC mixture exposure disrupted testicular gene expression of the prepubertal ram lambs. Affected genes included LHR, HSD3B1, HSD17B3 which are involved in the regulation of testosterone production and secretion (Barbagallo et al., 2020). The expression of markers of testosterone production has previously been shown to be sensitive to the effects of single ECs such as perfluorononanoic acid (PFNA) (Singh and Singh, 2019), di-isobutyl phthalate (Wang et al., 2017) and PFOS (Zhao et al., 2014). Further, we have previously, reported that EC mixture exposure results in higher levels of testicular HIF1α, which suppresses STAR and reduces testosterone biosynthesis (Elcombe et al., 2022b). In the current study BMP4 expression was also significantly affected by EC mixture exposure. BMP4 is important for establishing the blood-testis barrier and for germ cell differentiation and survival (Baleato et al., 2005; Li et al., 2014; Ma et al., 2023; Neumann et al., 2011; Wang et al., 2022). Finally, testicular ABP expression which is expressed in Sertoli cells and is crucial for maintaining the microenvironment for germ cells and supporting spermatogenesis, as well as facilitating sperm maturation in the epididymis was suppressed in the B group (Ma et al., 2015). A reduction in ABP expression following EC exposure has previously been reported in mice exposed to arsenic (Zeng et al., 2018) and PM2.5 (Ren et al., 2022). Surprisingly, the changes in testicular gene expression in the current study were not accompanied by altered mean testosterone levels. While the lack of an effect on testosterone could reflect the fact that the rams were prepubertal, it is possible that the higher expression of LHR, HSD3B1, HSD17B3, BMP4 in the B group may have been countered by the lower ABP expression so as to maintain normal testosterone concentrations. Alternatively, the observed changes in gene expression may be a compensatory process to offset an earlier onset of steroidogenesis as documented in a different subset of the same population of animals (Evans et al., 2023). In addition to effects on genes that could affect steroidogenesis and germ cell development, EC mixture exposure was also associated with increased testicular P27kip1 expression. P27kip1 is involved in the regulation of Sertoli cells maturation and proliferation and the observed changes in P27kip1 expression could inhibit Sertoli cells mitosis, and lead to aberrant spermatogenesis (Beumer et al., 1999; Yao et al., 2015). In this regard it is noteworthy that prepuberal B rams have fewer germ cells relative to Sertoli cells and more Sertoli-cell-only seminiferous tubules than C rams (Elcombe et al., 2022b). Since the ratio of Sertoli cells to germ cells in the testes is typically stable and species-specific (Jiang et al., 2019), the higher levels of P27kip1 expression in prepubertal testes may be a regulatory response to inhibit the further proliferation of Sertoli cells. CELF1 expression was also higher in the prepubertal B compared to C rams’ testis, it is potentially involved in various testicular functions from steroidogenesis to spermatogenesis regulation (Boulanger et al., 2015; Cibois et al., 2012). Mechanistically, it has been proposed that ECs’ endocrine disruption activity on the testis could be via oxidative stress which can impact on testicular steroidogenesis and affect LHR, HSD3B, HSD17B3, and CYP11A1 (Barbagallo et al., 2020; Mai et al., 2020; Santiago et al., 2021; Zhao et al., 2014). Studies of BPA-exposed adult rats (Doshi et al., 2011), have also suggested that ECs can affect the expression of various markers in tissues including testis through epigenetic routes. With regard to this later mechanism the expression of DNMT3a was higher in the testis of the prepubertal EC exposed males in the current study.

In contrast to the prepubertal animals, in whom prenatal EC exposure had no significant effects on hypothalamic gene expression, the adult B rams showed lower NKB, KISS1, and AR expression in the rostral ARC, and higher GNRH1 in the mPOA. These changes are in addition to our previously reported elevation of ESR1 expression in the same rostral ARC samples (Ghasemzadeh-Hasankolaei et al., 2023). These EC induced changes could be functionally important given that the ARC KNDy neurons expressing ESR1 are key regulators of GnRH secretion (Argente et al., 2023; Billings et al., 2010; Chimento et al., 2014; Uenoyama et al., 2021). The observed changes in KISS1 and ESR1 are consistent with those previously reported in the ARC of foetal B rams (Bellingham et al., 2009; Bellingham et al., 2016). The results are also consistent with the findings in in-utero DEHP exposed adult male rats in which hypothalamic NKB, ESR1, and KISS1 expression levels were downregulated (Gao et al., 2018). However, unlike our prior studies in foetal males in the biosolids exposure model (Bellingham et al., 2009; Bellingham et al., 2016) and DEHP exposed rats (Gao et al., 2018), GnRH expression in the adult B rams in the current study was increased despite reduced NKB and KISS1 expression. The differences between the current findings and those from our earlier studies could be due to the age/reproductive state of the animals, a possibility supported by the lack of an effect in the prepubertal animals in this study. EC exposure in the adult B rams was associated with decreased AR transcript levels in the rostral ARC and DLK1 in both the rostral and caudal ARC. AR is assumed to play important roles in regulating reproductive function and male sexual behaviour (Brock et al., 2015; Morford and Mauvais-Jarvis, 2016; Walters et al., 2018) whereas DLK1, codes for a preadipocyte factor, which may link metabolism and reproduction, the timing of puberty and the regulation of GnRH secretion (Argente et al., 2023; Gomes et al., 2019; Macedo and Kaiser, 2019) and thus could have contributed to the reproductive phenotype of this cohort of animals (Evans et al., 2023).

Developmental mixed EC exposure resulted in increased HSD3B1 and CYP11A1 expression within the adult testes, effects similar to those seen in in adult male rats exposed to DEHP in-utero (Culty et al., 2008) and DEHP (Qin et al., 2018). However, other studies have reported decreased testicular HSD3B and HSD17B mRNA and proteins in rats (Mai et al., 2020) and mice (Wang et al., 2017) developmentally exposed to TCDD and Di-isobutyl phthalate, respectively. The differences between the effects reported in these studies is most probably due to differences in the mechanisms of action of the individual ECs, with BTP exposure representing the combined effects from a complex mixture of ECs.

The ability to categorize prepubertal and adult biosolids-exposed animals into subgroups, that differed from each other and the controls, supports our previous findings of individual susceptibility to developmental EC mixture exposure in neo-natal [41] and adult [61] males. In the prepubertal rams, the one subgroup (8wBS1) the separation was based on several testicular markers related to germ, Sertoli and Leydig cells, and lower expression of GnRH and ESR1 in the mPOA. This subgroup therefore showed some shared changes particularly with regard to the downregulation of GnRH reported previously for male foetal sheep exposed to biosolids ECs in utero (Bellingham et al., 2016). The other subgroup (8wBS2) showed changes in three testicular markers and DNMT3a, with elevated pituitary KISSR mRNA levels compared to C rams. This group may represent a developmentally programmed effect, as GPR54 (KISS1R) expression was unaffected in previous studies of foetal lambs developmentally exposed to the EC mixture in biosolids (Bellingham et al., 2009). The elevated pituitary KISSR expression in this subgroup could have been driven by increased hypothalamic KISS1 and upregulated testicular steroidogenesis as evidenced by the higher expression of HSD3B1 and HSD17B3 (Yeung et al., 2011). Comparing the two B subgroups, it was evident that the EC induced changes in the 8wBS1 rams were distinct from 8wBS2 rams at all three levels of the HPG axis, and included difference is testicular FKBP6 and CIB1, both of which are crucial for male fertility and spermatogenesis (Wyrwoll et al., 2022; Yuan et al., 2006).

In the adults, the subgroups were defined by differences in the markers in the rostral ARC and mPOA (AdultBS1), while the other (AdultBS2) showed differences in the rostral ARC and testis. The subgroups also differed in ESR1 expression in both mentioned hypothalamic regions, a key testicular steroidogenesis marker, and blood testosterone concentrations. These findings suggest that the organizational consequences of in-utero exposure to biosolids ECs on the reproductive regulatory circuit, particularly in the hypothalamus and testis, can be observed during adulthood.

Comparing 8w and adult C rams, and 8w and adult B rams, revealed distinct changes in the expression patterns of 10 reproductive regulatory markers as they transitioned from the prepubertal to adult stage. These age/stage dependent gene alterations; 4 in the hypothalamus, 2 in the pituitary, and 4 in the testis, are likely to reflect the activation of the HPG axis (Baleato et al., 2005; Oyola and Handa, 2017). The analysis, however, also showed some changes that were unique to either B or C rams, and which therefore may represent either the loss or gain of changes as a consequence of B exposure. These findings undeniably demonstrate that preconceptional and in-utero exposure to the EC mixture from biosolids significantly impacted normal gene expression patterns within the HPG axis during the developmental transition from prepuberty to adulthood. A limitation of this study is that the exact chemical composition of the biosolids and the concentration of each specific chemical within biosolids remain unknown. It is therefore not possible to identify which specific chemical(s) in the biosolids are responsible for any observed effects. The exposure paradigm, of variable mixed EC exposure, however, does reflect what occurs due to environmental contamination and we have seen repeated effects across different cohorts of aniamls using the biosolid treated pasture model (add some refs you have already used from past studies).

In summary, data presented in this study clearly demonstrate that (1) preconceptional and in-utero exposure to the biosolids EC mixture had a significant impact on all three components of the HPG axis and (2) this is seen in both prepubertal and adult male offspring. Effects were observed at key genes mRNA and blood testosterone levels and were associated with phenotypical changes previously reported in this model. Future investigations could analyse changes in the transcriptome, proteome and epigenome of different parts of the HPG axis to provide clearer insights into the precise mechanisms through which EC mixtures influence various body tissues. The insights uncovered by this study constitute a key advancement in elucidating the intricate impact of EC exposure on tissue development and fundamental physiological processes such as reproduction and metabolism. This newfound understanding not only lays a foundation for exploring strategies to mitigate adverse effects of EC exposure but also holds immense promise in guiding interventions aimed at safeguarding public health from its potential consequences.

Supplementary Material

Supplementary information

Appendix A. Supplementary data

The Supplementary Information to this article is available online at:

Acknowledgments

The authors express their gratitude to Lynne Fleming and Dr Ana Monteiro for their invaluable assistance with laboratory procedures. We express our deep gratitude to the Cochno Farm and Research Centre staff for their invaluable technical assistance. Graphical abstract was created with BioRender.com.

Funding

This study was supported by a grant from the National Institutes of Health, USA (R01 ES030374).

Footnotes

Declaration of competing interest

None.

Ethical Approval

The study was conducted on sheep model at the University of Glasgow in accordance with the Home Office Animal (Scientific Procedures) Act (A(SP)A) 1986, under project licence PPL PF10145DF.

CRediT Authorship Contribution statement

Mohammad Ghasemzadeh-Hasankolaei: Investigation, Methodology, Writing- Original draft, Data collection and curation, Formal analysis. Neil P. Evans: Conceptualization, Investigation, Methodology, Writing- Original draft, Review & Editing, Funding acquisition, supervision, Project administration. Chris S. Elcombe: Review & Editing, Validation. Richard G. Lea: Conceptualization, Methodology, Review & Editing, Funding acquisition. Kevin D. Sinclair: Conceptualization, Methodology, Review & Editing. Vasantha Padmanabhan: Conceptualization, Methodology, Writing- Original draft, Review & Editing, Funding acquisition. Michelle Bellingham: Conceptualization, Methodology, Writing- Original draft, Review & Editing, supervision, Project administration.

Data availability

Data will be made available on request.

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Supplementary Materials

Supplementary information
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