Sex-specific prenatal stress effects on the rat reproductive axis and adrenal gland structure
Identifieur interne : 000426 ( Pmc/Corpus ); précédent : 000425; suivant : 000427Sex-specific prenatal stress effects on the rat reproductive axis and adrenal gland structure
Auteurs : Cheryl J. Ashworth ; Susan O. George ; Charis O. Hogg ; Yu-Ting Lai ; Paula J. BruntonSource :
- Reproduction (Cambridge, England) [ 1470-1626 ] ; 2016.
Abstract
Social stress during pregnancy has profound effects on offspring physiology. This
study examined whether an ethologically relevant social stress during late pregnancy
in rats alters the reproductive axis and adrenal gland structure in post-pubertal
male and female offspring. Prenatally stressed (PNS) pregnant rats
(
Url:
DOI: 10.1530/REP-16-0097
PubMed: 27026714
PubMed Central: 5065086
Links to Exploration step
PMC:5065086Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Sex-specific prenatal stress effects on the rat reproductive axis and
adrenal gland structure</title><author><name sortKey="Ashworth, Cheryl J" sort="Ashworth, Cheryl J" uniqKey="Ashworth C" first="Cheryl J" last="Ashworth">Cheryl J. Ashworth</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="George, Susan O" sort="George, Susan O" uniqKey="George S" first="Susan O" last="George">Susan O. George</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Hogg, Charis O" sort="Hogg, Charis O" uniqKey="Hogg C" first="Charis O" last="Hogg">Charis O. Hogg</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lai, Yu Ting" sort="Lai, Yu Ting" uniqKey="Lai Y" first="Yu-Ting" last="Lai">Yu-Ting Lai</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Brunton, Paula J" sort="Brunton, Paula J" uniqKey="Brunton P" first="Paula J" last="Brunton">Paula J. Brunton</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">27026714</idno><idno type="pmc">5065086</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5065086</idno><idno type="RBID">PMC:5065086</idno><idno type="doi">10.1530/REP-16-0097</idno><date when="2016">2016</date><idno type="wicri:Area/Pmc/Corpus">000426</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Sex-specific prenatal stress effects on the rat reproductive axis and
adrenal gland structure</title><author><name sortKey="Ashworth, Cheryl J" sort="Ashworth, Cheryl J" uniqKey="Ashworth C" first="Cheryl J" last="Ashworth">Cheryl J. Ashworth</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="George, Susan O" sort="George, Susan O" uniqKey="George S" first="Susan O" last="George">Susan O. George</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Hogg, Charis O" sort="Hogg, Charis O" uniqKey="Hogg C" first="Charis O" last="Hogg">Charis O. Hogg</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lai, Yu Ting" sort="Lai, Yu Ting" uniqKey="Lai Y" first="Yu-Ting" last="Lai">Yu-Ting Lai</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Brunton, Paula J" sort="Brunton, Paula J" uniqKey="Brunton P" first="Paula J" last="Brunton">Paula J. Brunton</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">Reproduction (Cambridge, England)</title><idno type="ISSN">1470-1626</idno><idno type="eISSN">1741-7899</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Abstract</title><p>Social stress during pregnancy has profound effects on offspring physiology. This
study examined whether an ethologically relevant social stress during late pregnancy
in rats alters the reproductive axis and adrenal gland structure in post-pubertal
male and female offspring. Prenatally stressed (PNS) pregnant rats
(<italic>n</italic>=9) were exposed to an unfamiliar lactating rat for 10 min/day
from day 16 to 20 of pregnancy inclusive, whereas control pregnant rats
(<italic>n</italic>=9) remained in their home cages. Gonads, adrenal glands and
blood samples were obtained from one female and one male from each litter at 11 to
12-weeks of age. Anogenital distance was measured. There was no treatment effect on
body, adrenal or gonad weight at 11–12 weeks. PNS did not affect the number of
primordial, secondary or tertiary ovarian follicles, numbers of corpora lutea or
ovarian FSH receptor expression. There was an indication that PNS females had more
primary follicles and greater ovarian aromatase expression compared with control
females (both <italic>P</italic>=0.09). PNS males had longer anogenital distances
(0.01±0.0 cm/g vs 0.008±0.00 cm/g; <italic>P</italic>=0.007) and higher
plasma FSH concentrations (0.05 ng/mL vs 0.006 ng/mL; s.e.d.=0.023;
<italic>P</italic>=0.043) compared with control males. There were no treatment
effects on the number of Sertoli cells or seminiferous tubules, seminiferous tubule
area, plasma testosterone concentration or testis expression of aromatase, FSH
receptor or androgen receptor. PNS did not affect adrenal size. These data suggest
that the developing male reproductive axis is more sensitive to maternal stress and
that PNS may enhance aspects of male reproductive development.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Abel, Mh" uniqKey="Abel M">MH Abel</name></author><author><name sortKey="Baker, Pj" uniqKey="Baker P">PJ Baker</name></author><author><name sortKey="Charlton, Hm" uniqKey="Charlton H">HM Charlton</name></author><author><name sortKey="Monteiro, A" uniqKey="Monteiro A">A Monteiro</name></author><author><name sortKey="Verhoeven, G" uniqKey="Verhoeven G">G Verhoeven</name></author><author><name sortKey="De Gendt, K" uniqKey="De Gendt K">K De Gendt</name></author><author><name sortKey="Guillou, F" uniqKey="Guillou F">F Guillou</name></author><author><name sortKey="O Haughnessy, Pj" uniqKey="O Haughnessy P">PJ O’Shaughnessy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Aitken, Ce" uniqKey="Aitken C">CE Aitken</name></author><author><name sortKey="Ozanne, Se" uniqKey="Ozanne S">SE 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Reproduction</journal-id><journal-id journal-id-type="iso-abbrev">Reproduction</journal-id><journal-id journal-id-type="hwp">REP</journal-id><journal-title-group><journal-title>Reproduction (Cambridge, England)</journal-title></journal-title-group><issn pub-type="ppub">1470-1626</issn><issn pub-type="epub">1741-7899</issn><publisher><publisher-name>Bioscientifica Ltd</publisher-name><publisher-loc>Bristol</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">27026714</article-id><article-id pub-id-type="pmc">5065086</article-id><article-id pub-id-type="doi">10.1530/REP-16-0097</article-id><article-id pub-id-type="publisher-id">REP160097</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research</subject></subj-group></article-categories><title-group><article-title>Sex-specific prenatal stress effects on the rat reproductive axis and
adrenal gland structure</article-title><alt-title alt-title-type="short">Reproduction in prenatally stressed rats</alt-title><alt-title alt-title-type="lrh">C J Ashworth and others<italic></italic></alt-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><name><surname>Ashworth</surname><given-names>Cheryl J</given-names></name><xref ref-type="aff" rid="aff1"></xref><xref ref-type="corresp" rid="cor1"></xref></contrib><contrib contrib-type="author"><name><surname>George</surname><given-names>Susan O</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Hogg</surname><given-names>Charis O</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Lai</surname><given-names>Yu-Ting</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Brunton</surname><given-names>Paula J</given-names></name><xref ref-type="aff" rid="aff1"></xref></contrib><aff id="aff1"><institution>The Roslin Institute and Royal (Dick) School of Veterinary Studies</institution><addr-line>University of Edinburgh, Edinburgh, UK</addr-line></aff></contrib-group><author-notes><corresp id="cor1">Correspondence should be addressed to C J Ashworth; Email:
<email>cheryl.ashworth@roslin.ed.ac.uk</email></corresp></author-notes><pub-date pub-type="ppub"><month>6</month><year>2016</year></pub-date><pub-date pub-type="epub"><month>6</month><year>2016</year></pub-date><volume>151</volume><issue>6</issue><fpage>709</fpage><lpage>717</lpage><history><date date-type="received"><day>4</day><month>8</month><year>2015</year></date><date date-type="rev-recd"><day>25</day><month>2</month><year>2016</year></date><date date-type="accepted"><day>29</day><month>3</month><year>2016</year></date></history><permissions><copyright-statement>© 2016 The authors</copyright-statement><copyright-year>2016</copyright-year><copyright-holder>The authors</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/3.0/deed.en_GB"><license-p><inline-graphic xlink:href="88x31.jpg"></inline-graphic>This
work is licensed under a <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/deed.en_GB">Creative
Commons Attribution 3.0 Unported License</ext-link>.</license-p></license></permissions><self-uri content-type="pdf" xlink:type="simple" xlink:href="rep-151-709.pdf"></self-uri><abstract><title>Abstract</title><p>Social stress during pregnancy has profound effects on offspring physiology. This
study examined whether an ethologically relevant social stress during late pregnancy
in rats alters the reproductive axis and adrenal gland structure in post-pubertal
male and female offspring. Prenatally stressed (PNS) pregnant rats
(<italic>n</italic>=9) were exposed to an unfamiliar lactating rat for 10 min/day
from day 16 to 20 of pregnancy inclusive, whereas control pregnant rats
(<italic>n</italic>=9) remained in their home cages. Gonads, adrenal glands and
blood samples were obtained from one female and one male from each litter at 11 to
12-weeks of age. Anogenital distance was measured. There was no treatment effect on
body, adrenal or gonad weight at 11–12 weeks. PNS did not affect the number of
primordial, secondary or tertiary ovarian follicles, numbers of corpora lutea or
ovarian FSH receptor expression. There was an indication that PNS females had more
primary follicles and greater ovarian aromatase expression compared with control
females (both <italic>P</italic>=0.09). PNS males had longer anogenital distances
(0.01±0.0 cm/g vs 0.008±0.00 cm/g; <italic>P</italic>=0.007) and higher
plasma FSH concentrations (0.05 ng/mL vs 0.006 ng/mL; s.e.d.=0.023;
<italic>P</italic>=0.043) compared with control males. There were no treatment
effects on the number of Sertoli cells or seminiferous tubules, seminiferous tubule
area, plasma testosterone concentration or testis expression of aromatase, FSH
receptor or androgen receptor. PNS did not affect adrenal size. These data suggest
that the developing male reproductive axis is more sensitive to maternal stress and
that PNS may enhance aspects of male reproductive development.</p></abstract></article-meta></front><body><sec><title>Introduction</title><p>There is a wealth of epidemiological and experimental data showing that the environment
to which a pregnant female is exposed can affect a range of physiological systems in the
offspring, including reproductive development (<xref rid="bib11" ref-type="bibr">Davies
& Norman 2002</xref>). Exposure to stressful prenatal environments, including
stressors such as extremes of temperature, light intensity, stocking density or
restraint or social stressors such as mixing with unfamiliar, older or aggressive
animals, can have profound post-natal effects (<xref rid="bib40" ref-type="bibr">Ward
& Weisz 1984</xref>, <xref rid="bib25" ref-type="bibr">Meek et al.
2006</xref>, <xref rid="bib35" ref-type="bibr">Schöpper et al. 2012</xref>, <xref rid="bib31" ref-type="bibr">Pallarés et al. 2013</xref>). Of particular
relevance and current interest are the prenatal effects of ethologically relevant
stressors, such as social stress, which better reflect real-life experiences a pregnant
female may encounter (for review see <xref rid="bib8" ref-type="bibr">Brunton
2013</xref>), on offspring viability and development.</p><p>Studies conducted in a range of mammalian species have demonstrated that prenatal stress
affects many aspects of reproduction including sexual differentiation of the brain,
development of the reproductive organs, puberty onset, reproductive behaviour, gonad
function and concentrations of reproductive hormones. The diverse prenatal stress
paradigms studied, the range of reproductive end points measured and the variation in
reproductive maturity of the offspring assessed in these studies make it challenging to
reach any consensus. One of the more frequent conclusions, based on the effects of
prenatal stress on reproductive behaviour (<xref rid="bib42" ref-type="bibr">Weinstock
2001</xref>, <xref rid="bib12" ref-type="bibr">Gerardin et al. 2005</xref>, <xref rid="bib38" ref-type="bibr">Wang et al. 2006</xref>), anogenital distance (<xref rid="bib20" ref-type="bibr">Lay et al. 2008</xref>, <xref rid="bib31" ref-type="bibr">Pallarés et al. 2013</xref>), reproductive hormone concentrations (<xref rid="bib39" ref-type="bibr">Ward & Weisz 1980</xref>, <xref rid="bib3" ref-type="bibr">Ashworth et al. 2011</xref>, <xref rid="bib31" ref-type="bibr">Pallarés et al. 2013</xref>) and both central neurotransmitter profiles
(<xref rid="bib6" ref-type="bibr">Bowman et al. 2004</xref>) and gene expression
(<xref rid="bib27" ref-type="bibr">Morgan & Bale 2011</xref>), is that
prenatal stress de-masculinises or feminises the male offspring. There is also some
evidence that prenatal stress masculinises the female offspring (e.g. <xref rid="bib17" ref-type="bibr">Kaiser et al. 2003</xref>, <xref rid="bib6" ref-type="bibr">Bowman et al. 2004</xref>), suggesting that, in general, prenatal stress
reduces the difference between male and female reproductive behaviour and physiology in
the offspring.</p><p>A recent re-analysis of published data describing sex differences in responses of human
foetuses to adverse maternal environments supported the widely held view that developing
male foetuses are more susceptible to changes in the maternal environment than female
foetuses (<xref rid="bib34" ref-type="bibr">Sandman et al. 2013</xref>). Furthermore,
evidence from litter-bearing animals indicates that the foetal male reproductive axis is
more sensitive to environmental change than the reproductive axis of female siblings
(rats: <xref rid="bib40" ref-type="bibr">Ward & Weisz 1984</xref>, pigs: <xref rid="bib3" ref-type="bibr">Ashworth et al. 2011</xref>). The reasons for such sex
differences are unknown, but may reflect higher growth rates in male foetuses (<xref rid="bib2" ref-type="bibr">Aitken & Ozanne 2013</xref>), sex differences in
the timing of the development of the foetal reproductive axis or in the need for
pituitary support of foetal gonad development (<xref rid="bib15" ref-type="bibr">Huhtaniemi 1989</xref>). Here, the aim is to determine whether sex differences in
the response of the reproductive system to an ethologically relevant social stress
imposed during pregnancy exist, as this could have important consequences for offspring
management and fertility.</p><p>Various mechanisms have been proposed to explain how the physiological consequences of
social stressors experienced by a pregnant female may be transmitted to her developing
foetuses including alterations in the opioid system (<xref rid="bib33" ref-type="bibr">Reznikov et al. 2005</xref>), epigenetic changes to placental gene expression (<xref rid="bib32" ref-type="bibr">Peña et al. 2012</xref>), alterations in the
maternal hypothalamic–pituitary–adrenal (HPA) axis and offspring HPA axis
responsiveness (reviewed by <xref rid="bib8" ref-type="bibr">Brunton 2013</xref>). It is
generally accepted that stress elevates maternal glucocorticoid levels with some studies
suggesting that the placenta of stressed mothers is less able to protect the foetus from
high glucocorticoid levels (<xref rid="bib23" ref-type="bibr">Mairesse et al.
2007</xref>) and that prenatally stressed offspring have enhanced HPA axis responses
to stress (<xref rid="bib7" ref-type="bibr">Brunton & Russell 2010</xref>).
Although thermal stress experienced immediately before sample collection alters the
structure of the rat adrenal cortex (<xref rid="bib19" ref-type="bibr">Koko et al.
2004</xref>), there appear to be few, if any, studies that determined whether the
structure of the adrenal gland can be programmed by prenatal stress. </p><p>The objectives of this study are (1) to conduct an integrated assessment of the
consequences of prenatal stress on reproductive status (hormone concentrations, gonad
histology and gene expression) and on adrenal gland size and structure and (2) to
determine whether any sex differences in the response exist. We hypothesised that, as
with our earlier studies in pigs, the developing male reproductive axis would be more
susceptible to prenatal stress and that the zona fasciculata of the adrenal cortex,
which produces corticosterone, may be a proportionally larger component of the adrenal
gland.</p></sec><sec><title>Materials and methods</title><sec><title>Experimental animals</title><p>All procedures were performed with the approval from the University of Edinburgh
Animal Welfare and Ethical Review Board and in accordance with the current UK Home
Office legislation. Sprague Dawley rats were purchased from Charles River (Margate,
Kent, UK). Rats were group housed (unless otherwise stated) in open-top cages and
maintained on a 12-h light:12-h darkness cycle (lights on at 07:00h) under controlled
conditions of temperature and humidity and with chow and water available <italic>ad
libitum</italic>. Pregnant rats were obtained by overnight mating with sexually
experienced males, and the presence of a vaginal semen plug the following morning was
designated as day 1 of pregnancy. All pregnant rats were housed individually from day
14 of pregnancy and weighed daily on days 16–20. After weighing, control rats
(C; <italic>n</italic>=9) were returned to their home cages, whereas stressed rats
(PNS; <italic>n</italic>=9) were exposed to social stress by transferring them into
the home cage of a different unfamiliar lactating rat (between days 2 and 9 of
lactation) for 10min/day on days 16–20 of pregnancy inclusive, as described
previously (<xref rid="bib7" ref-type="bibr">Brunton & Russell 2010</xref>).
This period of gestation coincides with foetal Sertoli cell proliferation (<xref rid="bib30" ref-type="bibr">Orth 1982</xref>) and adrenal gland differentiation
(<xref rid="bib26" ref-type="bibr">Mitani et al. 1997</xref>). Moreover, we have
demonstrated that this prenatal stress paradigm alters
hypothalamo–pituitary–adrenal axis function in the adult offspring
(<xref rid="bib7" ref-type="bibr">Brunton & Russell 2010</xref>). After day
20 of gestation, rats were undisturbed throughout the remaining period of pregnancy,
parturition and lactation (except for routine husbandry).</p><p>Pups were sexed on post-natal day 1 (PND1) and the combined weight of all the pups of
the same sex within a litter was recorded. Dams remained within their litters until
weaning on post-natal day 22. Weaned offspring were housed in groups by litter and
sex. At 81±0.44 days of age, one male and one female offspring were randomly
selected from each litter (to avoid within-litter effects), weighed, then killed by
conscious decapitation and anogenital distance measured. Trunk blood was collected
from each animal using 5% EDTA (Sigma-Aldrich) as an anti-coagulant, centrifuged at
1500 <bold><italic>g</italic></bold> for 10min at 4°C and the separated plasma
was stored at −20°C. Adrenal glands and gonads were rapidly removed,
surrounding fat was removed and the epididymis dissected from the testes and the
organs were weighed. One adrenal gland and one gonad from each animal were frozen in
liquid nitrogen and stored at −80°C, and the other adrenal gland and
gonad were placed in methacarn solution (60% methanol, 30% chloroform and 10% acetic
acid (Fisher Scientific)) for 24hours.</p></sec><sec><title>Histology</title><p>After 24hours, methacarn-fixed tissues were transferred to 100% ethanol (Fisher
Scientific) and stored at room temperature for 24hours. Samples were placed in
histological cassettes, dehydrated by passing through graded ethanol and xylene
(Fisher Scientific) and embedded in paraffin wax. From each specimen, 5 or 7µm
sections were cut and placed on Polysine microscope slides (Fisher Scientific).</p><sec><title>Ovarian tissue</title><p>The number of ovarian follicles and corpora lutea in ovarian sections was
estimated following immunohistochemical detection of proliferating cell nuclear
antigen (PCNA). Following dewaxing and heat-induced epitope retrieval (HIER) in
0.01M sodium citrate (Vector Laboratories), endogenous peroxidase activity and
non-specific binding sites were blocked by sequentially incubating slides with
0.3% H<sub>2</sub>O<sub>2</sub> (Sigma-Aldrich), in methanol and 1.5% normal horse
serum (Vectastain elite ABC kit; Vector Laboratories). Two 5µm-thick
sections, 100µm apart, from each ovary were incubated with mouse anti-rat
PCNA (Dako UK) at 1:800 dilution or with mouse IgG (Vector Laboratories) (at the
same total protein concentration) as the negative control. The slides were
incubated in a humidified chamber at room temperature for 30min followed by a
further 30min at room temperature with a biotinylated anti-mouse IgG secondary
antibody (Vectastain Elite ABC kit; Vector Laboratories) at a dilution of 1:200 in
1.5% normal horse serum. Sections were incubated with Vectastain elite ABC reagent
for 30min and with Novared peroxidase substrate (Vector Laboratories) for 5min.
They were then counterstained with haematoxylin and dehydrated in a graded series
of ethanol and xylene. Bright-field images were captured using a Nikon E600
microscope and a Scion cfw16012 colour camera and analysed using Image J
software.</p><p>Two PCNA-stained slides from each animal were examined in order to estimate the
number of primordial, transitory, primary, small pre-antral, large pre-antral and
Graafian follicles, categorised as described by <xref rid="bib22" ref-type="bibr">Lundy et al. (1999)</xref>, and the number of corpora lutea. The number of
large follicles and corpora lutea was counted at 1× magnification, whereas
the number of smaller follicles was counted at 20× magnification.</p></sec><sec><title>Testicular tissue</title><p>Slides of testis tissue were either stained with haematoxylin (VWR, Lutterworth,
UK) and eosin (Sigma-Aldrich) (H&E) or immunohistochemically stained with
GATA4 (Santa Cruz Biotechnology). Bright-field images were captured using a Nikon
E800XY and a Scion cfw16012 colour camera (H&E-stained slides) or E600
microscope and a Zeiss Axiocam 105 (GATA-4-stained slides). All images were
analysed using Image J software.</p><p>From H&E-stained slides, the mean external area of the seminiferous tubule
was determined by measuring ten seminiferous tubules per testis at 10×
magnification. The numbers of spermatogonia surrounding these seminiferous tubules
were determined. The number of seminiferous tubules was estimated by counting
tubules in two 4× magnification images from each of four pre-determined
sites on one testis slide from each animal. Spermatogenesis was categorised
according to Johnsen’s score for humans (<xref rid="bib16" ref-type="bibr">Johnsen 1970</xref>), which was modified for use in rat by <xref rid="bib21" ref-type="bibr">Lewis-Jones and Kerrigan (1985)</xref>. A mean
Johnsen’s score was calculated from ten seminiferous tubules per rat.</p><p>Following immunohistological detection of the Sertoli cell marker, GATA4, the
number of Sertoli cells in a defined area was determined. Dewaxing, epitope
retrieval and blocking were carried out in the same manner as for the ovaries. One
7µm section from each testis was incubated with rabbit anti-human GATA4 at
a dilution of 1:100 or with rabbit IgG (Vector Laboratories) at the same total
protein concentration as a negative control. The slides were incubated in a
humidified chamber at room temperature for 30min followed by a further 30min at
room temperature with a biotinylated anti-rabbit IgG secondary antibody
(Vectastain Elite ABC kit; Vector Laboratories) at a dilution of 1:200 in 1.5%
normal horse serum. Sections were incubated with Vectastain elite ABC reagent for
30min and with Novared peroxidase substrate (Vector Laboratories) for 10min. They
were then counterstained with haematoxylin and dehydrated in a graded series of
ethanol and xylene. Sertoli cells in five images of each testis were counted at
10× magnification and their numbers related to the area of the image.</p></sec><sec><title>Adrenal tissue</title><p>Sections (5µm) of adrenal tissue were stained with haematoxylin (VWR,
Lutterworth, UK) and eosin (Sigma-Aldrich). Bright-field images were captured
using a Nikon E600 microscope and a Scion cfw16012 colour camera and analysed
using Image J software. Three H&E-stained full adrenal cross sections per
animal were assessed. The total areas of the adrenal gland, adrenal medulla, zona
glomerulosa, zona fasciculate and zona reticularis of the adrenal cortex were
estimated at 20× magnification.</p></sec></sec><sec><title>Hormone concentrations</title><p>Plasma concentrations of follicle-stimulating hormone (FSH) and testosterone were
determined by ELISA using commercially available kits validated for rat samples (FSH:
USCN-Life, Wuhan, China; Testosterone: MP Biomedicals, Santa Ana, CA, USA). All
samples were analysed in a single assay. The minimum detectable concentrations were
0.0013pg/mL and 0.1ng/mL for FSH and testosterone respectively.</p></sec><sec><title>Expression of aromatase and androgen and FSH receptor</title><p>RNA was isolated from a maximum of 30mg frozen testis or ovarian tissue and cDNA
prepared as described by <xref rid="bib14" ref-type="bibr">Hernandez et al.
(2013)</xref>. The concentrations of the isolated RNA were determined
spectrophotometrically using a Nanodrop ND-1000 (Thermo Scientific). The RNA quality
was assessed either by a RNA Nano chip on an Agilent 2100 Bioanalyser (Agilent
Technologies) (testes) or by the High Sensitivity R6K ScreenTape System on an Agilent
2200 TapeStation (Agilent Technologies) (ovaries). The average RNA integrity number
for testes tissue was 9.66 (minimum 9.3), and the average RNA integrity number
equivalent for ovarian tissue was 8.93 (minimum 8.2).</p><p>The relative expression of androgen receptor (<italic>Ar</italic>) transcripts in
testis tissue and FSH receptor (<italic>Fshr</italic>) and aromatase
(<italic>Cyp19a1</italic>) transcripts in testis and ovarian tissue was measured
by qPCR in a Stratagene MX3005P Real Time PCR instrument (Agilent Technologies) using
Platinum SYBR Green Supermix UDG (Life Technologies) in accordance with the MIQE
guidelines (<xref rid="bib10" ref-type="bibr">Bustin et al. 2009</xref>). The primers
used are presented in <xref ref-type="table" rid="tbl1">Table 1</xref>. The aromatase
primers were designed using Primer-BLAST from NCBI. Previously published primers were
used for <italic>Ar</italic> (<xref rid="bib31" ref-type="bibr">Pallarés et
al. 2013</xref>) and <italic>Fshr</italic> (<xref rid="bib5" ref-type="bibr">Bernal et al. 2010</xref>). All gene-of-interest (GOI) primers were manufactured
by Life Technologies.<table-wrap id="tbl1" position="float"><label>Table 1</label><caption><p>Primer sequences used for qPCR.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"><bold>Gene</bold></th><th align="center" rowspan="1" colspan="1"><bold>Accession number</bold></th><th align="center" rowspan="1" colspan="1"><bold>Amplicon size</bold></th><th align="center" rowspan="1" colspan="1"><bold>Forward primer</bold></th><th align="center" rowspan="1" colspan="1"><bold>Reverse primer</bold></th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Aromatase (<italic>Cyp19a1</italic>)</td><td align="center" rowspan="1" colspan="1">M33986</td><td align="center" rowspan="1" colspan="1">303bp</td><td align="left" rowspan="1" colspan="1">TGC GTC CTA ACA TCA CTC TAC TG</td><td align="left" rowspan="1" colspan="1">AGT TGC AGG CAC TTC CA</td></tr><tr><td align="left" rowspan="1" colspan="1">Follicle-stimulating hormone receptor
(<italic>Fshr</italic>)</td><td align="center" rowspan="1" colspan="1">NM199237</td><td align="center" rowspan="1" colspan="1">151bp</td><td align="left" rowspan="1" colspan="1">TTG CTC CTG GTC TCC TTG CT</td><td align="left" rowspan="1" colspan="1">ACC TCA GTT CAA TGG CGT TCC</td></tr><tr><td align="left" rowspan="1" colspan="1">Androgen receptor (<italic>Ar</italic>)</td><td align="center" rowspan="1" colspan="1">NM012502.1</td><td align="center" rowspan="1" colspan="1">64bp</td><td align="left" rowspan="1" colspan="1">TCA CGC ACT GGC TGT ACA TTC</td><td align="left" rowspan="1" colspan="1">TGC ACC TGA CCT GGT TTT CA</td></tr></tbody></table></table-wrap></p><p>Reference genes were selected using a rat-specific geNorm reference gene selection
kit (PrimerDesign Ltd., Southampton, UK) following the manufacturer’s
protocol. <italic>Fshr</italic> in ovarian tissues, <italic>Ar</italic> in testis
tissues and <italic>Cyp19a1</italic> expression in both gonads were normalised using
β2-microglobulin (<italic>B2m</italic>) and ATP synthase subunit beta
(<italic>Atp5b</italic>). <italic>Fshr </italic>in testes was normalised using
<italic>B2m</italic> and glyceraldehyde 3 phosphate dehydrogenase
(<italic>Gapdh</italic>). cDNAs from all the samples were pooled and diluted from
1:5 to 1:5120 in nuclease-free water as standards. The cDNA of each sample and
reverse transcription control was diluted at 1:25 in RNase-free water. A total of
20µL master mix (12.5µL Platinum SYBR Green qPCR SuperMix UDG,
0.1µL ROX reference dye, 2µL of each GOI primer (400 nM final
concentration) or 1µL of each reference gene primer mix (240nM final
concentration), 3.4µL (or 6.4µL for reference genes) nuclease-free
water) was added to each well. Five microlitres of blanks (nuclease-free water), each
serial dilution of standard, the samples and reverse transcription control were
analysed in duplicate for each gene. PCR was carried out under the following
conditions: 2min at 50°C, 2min at 95°C followed by 40 cycles of 15s at
95°C and 30s at 60°C. A dissociation stage of 1min. at 95°C and
30s at 60°C followed by data collection between 60°C and the final 30
second 95°C step was included.</p><p>These data were analysed using qBase+ software (Biogazelle, Zwijnaarde,
Belgium) with target- and run-specific amplification efficiencies. The results were
normalised to two reference genes and expressed relative to either the minimum value
(<italic>Ar</italic> and <italic>Fshr</italic>) or a positive control
(<italic>Cyp19a1</italic>).</p></sec><sec><title>Statistical analysis</title><p>Individual pup daily weight gain between PND1 and the day of death was calculated as
was the paired adrenal and gonad weight expressed as a percentage of total body
weight and the anogenital distance:weight ratio. Effects of prenatal stress on litter
size, total litter weight, litter sex ratios, testis morphology, and gene expression
(males only) and ovarian morphology and gene expression (females only) were analysed
by one-way analysis of variance using Genstat (Version 13.1 for Windows; VSN
International Ltd., Oxford, UK). The effects of treatment, pup sex and their
interaction on birth weight, daily weight gain, weight at death, anogenital distance,
adrenal gland weight, areas of zones within the adrenal gland and hormone
concentrations were analysed by two-way analysis of variance. Some variables (plasma
FSH, female testosterone concentrations and ovarian aromatase expression) were log
transformed to overcome scale effects. In each case, <italic>P</italic><0.05
was considered to be statistically significant.</p></sec></sec><sec><title>Results</title><sec><title>Growth and organ size</title><p>Adrenal glands from one male PNS rat was not collected. Mean total litter size was
13.8±0.37 pups, with no differences between treatments. Within litters, 48.7%
of the pups were male. Sex ratios were unaffected by treatment. Total litter weight
on PND1 was significantly lower in the prenatally stressed pups compared with the
controls (70.23±1.43g vs 80.92±3.21g; <italic>P</italic>=0.005), with
both male and female prenatally stressed rats being lighter than controls of the same
sex (<xref ref-type="table" rid="tbl2">Table 2</xref>). There was no effect of
prenatal stress on post-natal daily weight gain, weight at cull, absolute anogenital
distance or relative adrenal gland or gonad weight (<xref ref-type="table" rid="tbl2">Table 2</xref>). Anogenital distance, expressed relative to body weight, was
significantly longer in PNS rats compared with control rats (0.0071 vs 0.0064,
s.e.d.=0.0003cm/g; <italic>P</italic>=0.021) respectively. There was also a
significant interaction (<italic>P</italic>=0.007), in which PNS reduced female
anogenital distance/g but increased anogenital distance/g in the male rats. As
expected, male rats grew more rapidly and were heavier at cull with heavier gonads
and longer anogenital distances than their female counterparts. However, the adrenal
glands accounted for a higher percentage of body weight in the female rats (<xref ref-type="table" rid="tbl2">Table 2</xref>). <table-wrap id="tbl2" position="float"><label>Table 2</label><caption><p>Body weight, weight change, anogenital distance and adrenal and gonad
weights in prenatally stressed and control rats.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" colspan="1"></th><th align="center" colspan="2" rowspan="1"><bold>Control</bold></th><th align="center" colspan="2" rowspan="1"><bold>Prenatally stressed</bold></th><th align="center" colspan="3" rowspan="1"><bold><italic>P</italic></bold></th></tr><tr><th align="center" rowspan="1" colspan="1">Female</th><th align="center" rowspan="1" colspan="1">Male</th><th align="center" rowspan="1" colspan="1">Female</th><th align="center" rowspan="1" colspan="1">Male</th><th align="center" rowspan="1" colspan="1">Treatment</th><th align="center" rowspan="1" colspan="1">Sex</th><th align="center" rowspan="1" colspan="1">Treatment × sex</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>n</italic></td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">Mean pup weight on PND1 (g)</td><td align="center" rowspan="1" colspan="1">5.81±0.35</td><td align="center" rowspan="1" colspan="1">6.15±0.38</td><td align="center" rowspan="1" colspan="1">5.03±0.21</td><td align="center" rowspan="1" colspan="1">5.32±0.23</td><td align="center" rowspan="1" colspan="1"><italic>P</italic>=0.011</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Weight at death (g)</td><td align="center" rowspan="1" colspan="1">225.16±4.04</td><td align="center" rowspan="1" colspan="1">343.9±22.79</td><td align="center" rowspan="1" colspan="1">215.29±7.84</td><td align="center" rowspan="1" colspan="1">330.38±11.97</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.001</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Weight gain between PND1 and death (g/day)</td><td align="center" rowspan="1" colspan="1">2.67±0.04</td><td align="center" rowspan="1" colspan="1">4.13±0.22</td><td align="center" rowspan="1" colspan="1">2.60±0.10</td><td align="center" rowspan="1" colspan="1">4.09±0.15</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.001</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Paired gonad weight as % body weight</td><td align="center" rowspan="1" colspan="1">0.08±0.003</td><td align="center" rowspan="1" colspan="1">1.01±0.06</td><td align="center" rowspan="1" colspan="1">0.08±0.003</td><td align="center" rowspan="1" colspan="1">1.034±0.032</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.001</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Paired adrenal weight as % body weight</td><td align="center" rowspan="1" colspan="1">0.040±0.002</td><td align="center" rowspan="1" colspan="1">0.023±0.001</td><td align="center" rowspan="1" colspan="1">0.038±0.002</td><td align="center" rowspan="1" colspan="1">0.022±0.002*</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.001</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Anogenital distance (cm)</td><td align="center" rowspan="1" colspan="1">1.03±0.05</td><td align="center" rowspan="1" colspan="1">2.85±0.22</td><td align="center" rowspan="1" colspan="1">0.94±0.05</td><td align="center" rowspan="1" colspan="1">3.23±0.07</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.001</td><td align="center" rowspan="1" colspan="1">0.065</td></tr><tr><td align="left" rowspan="1" colspan="1">Anogenital distance (cm/g)</td><td align="center" rowspan="1" colspan="1">0.0046±0.0002</td><td align="center" rowspan="1" colspan="1">0.0083±0.0003</td><td align="center" rowspan="1" colspan="1">0.0044±0.0003</td><td align="center" rowspan="1" colspan="1">0.010±0.0003</td><td align="center" rowspan="1" colspan="1"><italic>P</italic>=0.021</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.001</td><td align="center" rowspan="1" colspan="1">0.007</td></tr></tbody></table><table-wrap-foot><fn id="tbl-fn1"><p>NS, not significant.</p></fn><fn id="tbl-fn2"><p>*<italic>n</italic>=8.</p></fn></table-wrap-foot></table-wrap></p></sec><sec><title>Histology</title><sec><title>Ovaries</title><p>The majority of ovarian follicles present in all the rats studied were primordial
follicles. Prenatally stressed females tended to have more primary follicles
(<italic>P</italic>=0.09); however, prenatal stress did not affect the number
of the other types of ovarian follicles, the distribution of follicle type or the
number of corpora lutea (<xref ref-type="table" rid="tbl3">Table
3</xref>).<table-wrap id="tbl3" position="float"><label>Table 3</label><caption><p>Abundance of ovarian follicles and corpora lutea in ovaries from
prenatally stressed and control rats.</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"></th><th align="center" rowspan="1" colspan="1"><bold>Control</bold></th><th align="center" rowspan="1" colspan="1"><bold>Prenatally stressed</bold></th><th align="center" rowspan="1" colspan="1"><bold><italic>P</italic></bold></th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>n</italic></td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">Primordial follicles</td><td align="center" rowspan="1" colspan="1">36.56±3.59</td><td align="center" rowspan="1" colspan="1">45.78±10.09</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Transitory follicles</td><td align="center" rowspan="1" colspan="1">7.33±0.97</td><td align="center" rowspan="1" colspan="1">11.0±2.55</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Primary follicles</td><td align="center" rowspan="1" colspan="1">3.78±0.57</td><td align="center" rowspan="1" colspan="1">5.33±0.65</td><td align="center" rowspan="1" colspan="1">0.09</td></tr><tr><td align="left" rowspan="1" colspan="1">Small pre-antral follicles</td><td align="center" rowspan="1" colspan="1">0.67±0.33</td><td align="center" rowspan="1" colspan="1">1.11±0.35</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Large pre-antral follicles</td><td align="center" rowspan="1" colspan="1">1.78±0.43</td><td align="center" rowspan="1" colspan="1">1.67±0.37</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Large antral follicles</td><td align="center" rowspan="1" colspan="1">8.0±0.62</td><td align="center" rowspan="1" colspan="1">8.56±1.01</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Corpora lutea</td><td align="center" rowspan="1" colspan="1">11.56±1.44</td><td align="center" rowspan="1" colspan="1">11.67±1.41</td><td align="center" rowspan="1" colspan="1">NS</td></tr></tbody></table><table-wrap-foot><fn id="tbl-fn3"><p>Values are mean number of follicles or corpora lutea from two whole
ovary cross sections per animal.</p></fn><fn id="tbl-fn4"><p>NS, not significant.</p></fn></table-wrap-foot></table-wrap></p></sec><sec><title>Testes</title><p>There were no significant effects of treatment on testis histology, although there
was a tendency for PNS rats to have more spermatogonia (<xref ref-type="table" rid="tbl4">Table 4</xref>).<table-wrap id="tbl4" position="float"><label>Table 4</label><caption><p>Testis development in prenatally stressed and control rats.</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"></th><th align="center" rowspan="1" colspan="1"><bold>Control</bold></th><th align="center" rowspan="1" colspan="1"><bold>Prenatally stressed</bold></th><th align="center" rowspan="1" colspan="1"><bold><italic>P</italic></bold></th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>n</italic></td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">Number of spermatogonia*<sup>$</sup></td><td align="center" rowspan="1" colspan="1">36±2.45</td><td align="center" rowspan="1" colspan="1">42.33±2.81</td><td align="center" rowspan="1" colspan="1">0.109</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of Sertoli cells/mm<sup>2</sup></td><td align="center" rowspan="1" colspan="1">94.431±1.80</td><td align="center" rowspan="1" colspan="1">96.565±2.09</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Area of seminiferous tubule
(mm<sup>2</sup>)<sup>$</sup></td><td align="center" rowspan="1" colspan="1">1.65±0.05</td><td align="center" rowspan="1" colspan="1">1.72±0.06</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Johnsen’s score<sup>$</sup></td><td align="center" rowspan="1" colspan="1">8.33±0.37</td><td align="center" rowspan="1" colspan="1">8.0±.33</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of seminiferous
tubules<sup>§</sup></td><td align="center" rowspan="1" colspan="1">50±2.31</td><td align="center" rowspan="1" colspan="1">48.72±2.61</td><td align="center" rowspan="1" colspan="1">NS</td></tr></tbody></table><table-wrap-foot><fn id="tbl-fn5"><p>NS, not significant.</p></fn><fn id="tbl-fn6"><p>*Associated with a seminiferous tubule. Values are the means of
<sup>$</sup>ten or <sup>§</sup>four testis sections
per animal.</p></fn></table-wrap-foot></table-wrap></p></sec><sec><title>Adrenal glands</title><p>There was a positive correlation between adrenal gland weight and cross-sectional
surface area (<italic>P</italic><0.02). <xref ref-type="fig" rid="fig1">Figure 1</xref> shows a representative section of the rat adrenal gland,
indicating the zones measured. There was no effect of prenatal treatment on the
cross-sectional area of the adrenal gland or on the relative abundance of the zona
glomerulosa, zona fasciculata or zona reticularis (<xref ref-type="table" rid="tbl5">Table 5</xref>). Adrenal glands from female rats had a larger
cross-sectional area with relatively less zona glomerulosa and relatively more
zona reticularis (<xref ref-type="table" rid="tbl5">Table 5</xref>). There were no
significant treatment x sex interactions.<fig fig-type="figure" id="fig1" position="float"><label>Figure 1</label><caption><p>Representative section of a day 81 rat adrenal gland showing the zones
measured. Adrenal medulla (A), zona reticularis (B), zona fasciculate (C)
and zona glomerulosa (D). Scale bar=1mm.</p></caption><graphic xlink:href="rep-151-709-g001"></graphic></fig><table-wrap id="tbl5" position="float"><label>Table 5</label><caption><p>Area of adrenal gland and relative area of adrenal gland zones in
prenatally stressed and control rats.</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"></th><th align="center" colspan="2" rowspan="1"><bold>Control</bold></th><th align="center" colspan="2" rowspan="1"><bold>Prenatally stressed</bold></th><th align="center" colspan="2" rowspan="1"><bold><italic>P</italic></bold></th></tr><tr><th align="left" rowspan="1" colspan="1"></th><th align="center" rowspan="1" colspan="1">Female</th><th align="center" rowspan="1" colspan="1">Male</th><th align="center" rowspan="1" colspan="1">Female</th><th align="center" rowspan="1" colspan="1">Male</th><th align="center" rowspan="1" colspan="1">Treatment</th><th align="center" rowspan="1" colspan="1">Sex</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><italic>n</italic></td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1"></td><td align="center" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">Area of adrenal gland (mm<sup>2</sup>)</td><td align="center" rowspan="1" colspan="1">338.66±15.9</td><td align="center" rowspan="1" colspan="1">281.92±18.24</td><td align="center" rowspan="1" colspan="1">323.0±10.27</td><td align="center" rowspan="1" colspan="1">264.37±9.51</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.001</td></tr><tr><td align="left" rowspan="1" colspan="1">Area of zona granulosa as % total area</td><td align="center" rowspan="1" colspan="1">9.56±0.42</td><td align="center" rowspan="1" colspan="1">10.81±0.35</td><td align="center" rowspan="1" colspan="1">9.34±0.22</td><td align="center" rowspan="1" colspan="1">11.19±0.55</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.001</td></tr><tr><td align="left" rowspan="1" colspan="1">Area of zona fasciculata as % of total area</td><td align="center" rowspan="1" colspan="1">54.20±1.58</td><td align="center" rowspan="1" colspan="1">56.72±2.12</td><td align="center" rowspan="1" colspan="1">57.50±1.63</td><td align="center" rowspan="1" colspan="1">57.06±1.49</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr><tr><td align="left" rowspan="1" colspan="1">Area of zona reticularis as % of total area</td><td align="center" rowspan="1" colspan="1">23.59±1.19</td><td align="center" rowspan="1" colspan="1">19.13±1.52</td><td align="center" rowspan="1" colspan="1">21.94±1.19</td><td align="center" rowspan="1" colspan="1">19.46±0.89</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1"><italic>P</italic><0.01</td></tr><tr><td align="left" rowspan="1" colspan="1">Area of adrenal medulla as % of total area</td><td align="center" rowspan="1" colspan="1">12.28±0.76</td><td align="center" rowspan="1" colspan="1">13.29±0.89</td><td align="center" rowspan="1" colspan="1">11.28±0.89</td><td align="center" rowspan="1" colspan="1">12.71±0.93</td><td align="center" rowspan="1" colspan="1">NS</td><td align="center" rowspan="1" colspan="1">NS</td></tr></tbody></table><table-wrap-foot><fn id="tbl-fn7"><p>NS, not significant.</p></fn></table-wrap-foot></table-wrap></p></sec><sec><title>Hormone concentrations</title><p>A plasma sample from a control female rat was lost during processing. FSH was
undetectable in plasma from female rats. Male rats that were prenatally stressed
had significantly higher FSH concentrations than control rats (0.05ng/mL vs
0.006ng/mL (s.e.d.=0.023, <italic>P</italic>=0.043) respectively). There was no
significant difference in plasma testosterone concentrations between male (CON:
9.64±1.39ng/mL vs PNS: 9.71±1.73ng/mL) or female (CON:
0.41±0.05ng/mL vs PNS: 0.60±0.12ng/mL) control or PNS rats
respectively.</p></sec><sec><title>Receptor expression</title><p>There was no difference between <italic>Fshr</italic> expression (2.29±0.25
vs 1.87±0.13 relative units) in control and PNS rat ovaries respectively.
Similarly, prenatal stress did not affect testis expression of <italic>Ar</italic>(1.29±0.03 vs 1.27±0.06 relative units) or <italic>Fshr</italic>(0.86±0.02 vs 0.85±0.03 relative units) in control and PNS rats
respectively.</p></sec><sec><title>Aromatase expression</title><p>There was no effect of prenatal stress on <italic>Cyp19a1 </italic>gene expression
in testis tissue; however, ovaries from PNS females tended to have less
<italic>Cyp19a1</italic> expression than ovaries from control females
(0.547±0.10 vs 0.36±0.103 relative units;
<italic>P</italic>=0.095).</p></sec></sec></sec><sec><title>Discussion</title><p>This study has shown that exposure to an ethologically relevant social stress during the
last week of pregnancy in the rat affected the reproductive axis of the male, but not
the female, offspring. The observations that prenatally stressed male rats had longer
relative anogenital distances and higher plasma FSH concentrations join the growing body
of recent data (<xref rid="bib31" ref-type="bibr">Pallarés et al. 2013</xref>,
<xref rid="bib9" ref-type="bibr">Brunton et al. 2015</xref>) indicating that some
aspects of reproductive function are enhanced in sexually mature male rat offspring
whose mothers were subjected to stress during pregnancy. Similar findings have been
found in sheep, in which sexually mature male offspring born to mothers managed in poor
husbandry conditions had larger testes with larger seminiferous tubules (<xref rid="bib28" ref-type="bibr">Moutevelidi et al. 2015</xref>). These findings are in
marked contrast to reports that suggest prenatal stress de-masculinises the male
offspring (<xref rid="bib39" ref-type="bibr">Ward & Weisz 1980</xref>, <xref rid="bib42" ref-type="bibr">Weinstock 2001</xref>, <xref rid="bib6" ref-type="bibr">Bowman et al. 2004</xref>, <xref rid="bib12" ref-type="bibr">Gerardin et al.
2005</xref>, <xref rid="bib38" ref-type="bibr">Wang et al. 2006</xref>, <xref rid="bib27" ref-type="bibr">Morgan & Bale 2011</xref>, <xref rid="bib31" ref-type="bibr">Pallarés et al. 2013</xref>). The reason(s) for this different
response are not known; however, it is possible that variations in the stage of
pregnancy when the stress was imposed, the duration, predictability and severity of the
stress may alter later outcomes. For example, the work described by <xref rid="bib6" ref-type="bibr">Bowman et al. (2004)</xref> and <xref rid="bib31" ref-type="bibr">Pallarés et al. (2013)</xref> imposed a restraint stress three
times a day for the complete last week of pregnancy, whereas the studies of <xref rid="bib27" ref-type="bibr">Morgan & Bale (2011)</xref> involved subjecting
mice to a range of stressors during early pregnancy.</p><p>Most studies investigating the effects of prenatal stress on rat reproductive
development suggest that, as in this study, effects are more pronounced in the male
offspring. However, a recent study by <xref rid="bib4" ref-type="bibr">Barra et al.
(2014)</xref> reported that exposure to 4°C for 3h a day throughout pregnancy
in rats delayed ovarian follicular development on post-natal days 4 and 30, and delayed
the onset of puberty in the female offspring. This study, which used post-pubertal rats,
showed no significant alteration in the populations of ovarian follicles, although
ovaries from prenatally stressed female rats tended to have more primary follicles. The
tendency for ovaries from rats born to stressed mothers to have lower aromatase mRNA
expression suggests that the ability to synthesise oestradiol may be compromised in
these animals.</p><p>The sex differences observed in this study could reflect the more dynamic nature of
male, compared with female, foetal reproductive development at the stage of gestation
when the stress was imposed. Rat foetal testes develop rapidly during the last week of
gestation with testicular cords formed on day 16, differentiation of Leydig cells
occurring on day 17 and the first wave of Sertoli and Leydig cell proliferation
occurring during late foetal life (<xref rid="bib30" ref-type="bibr">Orth 1982</xref>).
Foetal testes secrete testosterone, which reaches a prenatal peak on days 18 and 19
(<xref rid="bib43" ref-type="bibr">Weisz & Ward 1980</xref>) and is important
for post-natal reproductive development. By contrast, female rat pups are born with
relatively immature ovaries (<xref rid="bib37" ref-type="bibr">Takagi et al.
2007</xref>) that are steroidogenically quiescent until the second week of life (<xref rid="bib44" ref-type="bibr">Weniger 1993</xref>).</p><p>Sex differences in reproductive axis responses to prenatal stress are often explained by
differential effects of stress-induced alterations in the foetal HPA axis on the male
and female foetal hypothalamic–pituitary–gonadal (HPG) axis (<xref rid="bib11" ref-type="bibr">Davies & Norman 2002</xref>, <xref rid="bib8" ref-type="bibr">Brunton 2013</xref>). However, the rat testis appears to be independent of
pituitary hormone support throughout gestation (reviewed by <xref rid="bib29" ref-type="bibr">O’Shaughnessy & Fowler 2011</xref>), whereas the
responsiveness of the rat ovary to LH does not appear until the end of the first week of
life, coincident with the onset of ovarian steroidogenesis (<xref rid="bib36" ref-type="bibr">Sokka et al. 1996</xref>), suggesting that alterations in the foetal HPG
axis are not likely to explain the sex differences observed.</p><p>It is generally accepted that prenatal stress programmes HPA axis responses (<xref rid="bib8" ref-type="bibr">Brunton 2013</xref>). An earlier experiment using the same
prenatal stress paradigm as in this study reported that both male and female prenatally
stressed rats challenged with immune challenge or restraint stress show markedly greater
ACTH and corticosterone secretion than the controls (<xref rid="bib7" ref-type="bibr">Brunton & Russell 2010</xref>). In another study, heat-stress imposed for an
hour immediately before tissue collection caused an increase in plasma ACTH and
corticosterone concentrations in adult rats, which was accompanied by a reduction in the
overall size of the adrenal gland and of the zona fasciculata in particular (<xref rid="bib19" ref-type="bibr">Koko et al. 2004</xref>). This raises the question of
whether prenatal stress could programme either adrenal gland size or the relative size
of distinct functional zones within the adrenal gland. This study does not show an
effect of prenatal stress on the size or the structure of the adrenal gland, suggesting
that prenatal stress-associated alterations in corticosterone secretion are not a
consequence of changes in adrenal gland structure. Rather, they appear to be a result of
increased excitatory drive to the HPA axis at the level of the hypothalamus (<xref rid="bib7" ref-type="bibr">Brunton & Russell 2010</xref>). This study
confirmed that post-pubertal female rats have larger adrenal glands than males (<xref rid="bib13" ref-type="bibr">Hatai 1913</xref>) and that sex differences in the size
of the zones of the adrenal cortex exist (<xref rid="bib24" ref-type="bibr">Majchrzak
& Malendowicz 1983</xref>). Compared with male rats, female rats in this study
had a smaller zona glomerulosa, the zone primarily responsible for mineralocorticoid
secretion, and a larger zona reticularis, which secretes sex steroids. The significance
of this finding remains to be elucidated.</p><p>Of particular interest in this study is that the prenatally stressed male rats had
longer anogenital distances and higher plasma FSH concentrations. The anogenital
distance reflects the migration of the genital orifice to the navel during foetal life,
which is controlled by foetal testosterone concentration (<xref rid="bib45" ref-type="bibr">Wilson & Davies 2007</xref>). Longer anogenital distances are
therefore likely to reflect higher foetal testosterone concentrations. Although foetal
testosterone levels were not measured in this study, other models of maternal stress in
rats show an attenuation of the prenatal foetal testosterone surge (<xref rid="bib41" ref-type="bibr">Ward et al. 2003</xref>) and, in contrast to the results
of this study, a reduction in post-natal anogenital distance (<xref rid="bib31" ref-type="bibr">Pallarés et al. 2013</xref>). Our previous studies using the same
experimental paradigm found higher concentrations of total (but not free) testosterone
in prenatally stressed male offspring (<xref rid="bib9" ref-type="bibr">Brunton et al.
2015</xref>). In this study, testosterone concentrations in offspring were not
significantly different between treatments. The reason for this discrepancy is not
clear, but may reflect differences in the source of the plasma (trunk blood vs venous
blood) between the two experiments.</p><p>The increased FSH concentrations in PNS rats suggest that their Sertoli cells would be
exposed to more FSH, which along with testosterone regulates Sertoli cell function,
inhibin secretion and, indirectly, spermatogenesis (<xref rid="bib1" ref-type="bibr">Abel et al. 2008</xref>). In support of this, and although not statistically
significant, the number of spermatogonia per seminiferous tubule in this study tended to
be greater in the prenatally stressed males. Furthermore, a recent study reported that
the male offspring of rats that were subjected to restraint stress three times a day
during the last week of pregnancy had accelerated spermatogenesis both pre- and
post-puberty (<xref rid="bib31" ref-type="bibr">Pallarés et al. 2013</xref>),
further supporting the concept that prenatal stress may promote male reproductive
development. The physiological significance of this remains to be elucidated; however,
it may reflect a strategy to increase the likelihood that offspring born in
disadvantaged environments are able to reproduce.</p><p>The mechanisms by which the foetal male reproductive axis responds to stress-induced
changes in maternal physiology are unclear, but could involve alterations in the
relationship between maternal and/or the foetal HPA and HPG axes, epigenetic factors
and/or alterations in opioids and neurosteroids and will likely depend on the duration
and the severity of the stress imposed. For example, rats subjected to chronic restraint
stress during the last 7–11 days of pregnancy have reduced placental expression
and activity of the enzyme 11β-hydroxysteroid dehydrogenase type 2
(11βHSD2) (<xref rid="bib23" ref-type="bibr">Mairesse et al. 2007</xref>, <xref rid="bib32" ref-type="bibr">Peña et al. 2012</xref>), which protects the
foetus from high circulating concentrations of maternal corticosterone. <xref rid="bib32" ref-type="bibr">Peña et al. (2012)</xref> also report prenatal
stress-induced changes in DNA methylation at CpG sites within the placental and foetal
hypothalamic <italic>Hsd11b2</italic> gene promoter on embryonic day 20 and propose
alterations in DNA methylation as a mechanism by which prenatal stress alters placental
<italic>Hsd11b2</italic> expression.</p><p>In the study of <xref rid="bib23" ref-type="bibr">Mairesse et al. (2007)</xref>, day 21
foetuses carried by stressed dams did not have altered plasma corticosterone levels, but
did have atrophied adrenal glands and reduced foetal ACTH, suggesting that maternally
derived corticosterone was the major contributor to foetal concentrations. Male foetuses
also had smaller testes; however, there was no direct evidence that this was linked to
alterations in foetal or maternal glucocorticoids. Furthermore, <xref rid="bib7" ref-type="bibr">Brunton and Russell (2010)</xref> observed no effects of prenatal stress
on basal ACTH and corticosterone concentrations in 11-week-old male rat offspring. These
findings, coupled with the fact that foetal rat testis (<xref rid="bib29" ref-type="bibr">O’Shaughnessy & Fowler 2011</xref>) and ovary development
(<xref rid="bib36" ref-type="bibr">Sokka et al. 1996</xref>) is independent of
pituitary gonadotrophins, suggesting that alterations in the foetal HPG axis are not
likely to explain the response observed.</p><p>Other potential mechanisms should be considered. For example, observations that
administration of naltrexone to stressed pregnant rats blocks the feminising effects of
prenatal stress on male offspring (<xref rid="bib18" ref-type="bibr">Keshet &
Weinstock 1995</xref>, <xref rid="bib33" ref-type="bibr">Reznikov et al. 2005</xref>)
indicate that opioids may be involved in mediating the effects of stress on the
reproductive axis.</p><p>In conclusion, the results of this study indicate that exposure to an ethologically
relevant social stress may enhance the development of the reproductive axis in the male
offspring. Further studies are required to determine whether such an intervention is
associated with enhanced male reproductive performance.</p></sec><sec><title>Declaration of interest</title><p>The authors declare that there is no conflict of interest that could be perceived as
prejudicing the impartiality of the research reported.</p></sec><sec><title>Funding</title><p>The Roslin Institute receives Institute Strategic Grant funding from the BBSRC
(BB/J004316/1). Y T L was in receipt of a studentship from the Ministry of Education,
Taiwan.</p></sec></body><back><ack><title>Acknowledgements</title><p>The authors thank the staff of the Biological Resources Facility at the Roslin Institute
for Animal Care, Bob Fleming (Roslin Institute) for assistance with bioimaging and MSc
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