Peripheral and central mechanisms involved in hormonal control of male and female reproduction

Abstract

Reproduction involves the integration of hormonal signals acting across multiple systems to generate a synchronized physiological output. A critical component of reproduction is the luteinizing hormone (LH) surge, which is mediated by estradiol (E2) and neuroprogesterone interacting to stimulate kisspeptin release in the rostral periventricular nucleus of the third ventricle in rats. Recent evidence has shown that both classical and membrane E2 and progesterone signaling is involved in this pathway. A metabolite of gonadotropin-releasing hormone (GnRH), GnRH-(1-5), has been shown to stimulate GnRH expression, secretion, and has a role in the regulation of lordosis. Additionally, gonadotropin-inhibitory hormone (GnIH) projects to and influences the activity of GnRH neurons in birds. Stress-induced changes in GnIH have been shown to alter breeding behaviors in birds, demonstrating another molecular control of reproduction. Peripherally, paracrine and autocrine actions within the gonad have been suggested as therapeutic targets for infertility in both males and females. Dysfunction of testicular prostaglandin synthesis is a possible cause of idiopathic male infertility. Indeed, local production of melatonin and corticotropin-releasing hormone (CRH) could influence spermatogenesis via immune pathways in the gonad. In females, vascular endothelial growth factor A (VEGF-A) has been implicated in an angiogenic process that mediates development of the corpus luteum and thus fertility via the Notch signaling pathway. Age-induced decreases in fertility involve ovarian kisspeptin and its regulation of ovarian sympathetic innervation. Finally, morphological changes in the arcuate nucleus of the hypothalamus influence female sexual receptivity in rats. The processes mediating these morphological changes have been shown to involve rapid effects of E2 controlling synaptogenesis in this hypothalamic nucleus. Together, this review highlights new research in these areas, focusing on recent findings in the molecular mechanisms of central and peripheral hormonal control of reproduction. This article is protected by copyright. All rights reserved.

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Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12405
REVIEW ARTICLE ©2016 British Society for Neuroendocrinology
Peripheral and Central Mechanisms Involved in the Hormonal Control of
Male and Female Reproduction
L. M. Rudolph*, G. E. Bentley†, R. S. Calandra‡, A. H. Paredes§, M. Tesone‡,T.J.Wu¶and P. E. Micevych*
*Department of Neurobiology, Laboratory of Neuroendocrinology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
†Department of Integrative Biology, and Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA.
‡Instituto de Biolog

ıa y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina.
§Laboratory of Neurobiochemistry, Faculty of Chemistry and Pharmaceutical Sciences, Universidad de Chile, Independencia, Santiago, Chile.
¶Department of Obstetrics and Gynecology, Center for Neuroscience and Regenerative Medicine, Uniformed Services University, Bethesda, MD, USA.
Journal of
Neuroendocrinology
Correspondence to: Lauren M.
Rudolph, UCLA Department of
Neurobiology, CHS 73-074, 650
Charles E. Young Dr. S., Los Angeles,
CA 90095, USA (e-mail:
lrudolph@mednet.ucla.edu).
Reproduction involves the integration of hormonal signals acting across multiple systems to
generate a synchronised physiological output. A critical component of reproduction is the lutein-
ising hormone (LH) surge, which is mediated by oestradiol (E
2
) and neuroprogesterone interact-
ing to stimulate kisspeptin release in the rostral periventricular nucleus of the third ventricle in
rats. Recent evidence indicates the involvement of both classical and membrane E
2
and proges-
terone signalling in this pathway. A metabolite of gonadotrophin-releasing hormone (GnRH),
GnRH-(1-5), has been shown to stimulate GnRH expression and secretion, and has a role in the
regulation of lordosis. Additionally, gonadotrophin release-inhibitory hormone (GnIH) projects to
and influences the activity of GnRH neurones in birds. Stress-induced changes in GnIH have
been shown to alter breeding behaviour in birds, demonstrating another mechanism for the
molecular control of reproduction. Peripherally, paracrine and autocrine actions within the
gonad have been suggested as therapeutic targets for infertility in both males and females. Dys-
function of testicular prostaglandin synthesis is a possible cause of idiopathic male infertility.
Indeed, local production of melatonin and corticotrophin-releasing hormone could influence
spermatogenesis via immune pathways in the gonad. In females, vascular endothelial growth
factor A has been implicated in an angiogenic process that mediates development of the corpus
luteum and thus fertility via the Notch signalling pathway. Age-induced decreases in fertility
involve ovarian kisspeptin and its regulation of ovarian sympathetic innervation. Finally, mor-
phological changes in the arcuate nucleus of the hypothalamus influence female sexual recep-
tivity in rats. The processes mediating these morphological changes have been shown to involve
the rapid effects of E
2
controlling synaptogenesis in this hypothalamic nucleus. In summary, this
review highlights new research in these areas, focusing on recent findings concerning the
molecular mechanisms involved in the central and peripheral hormonal control of reproduction.
Key words: progesterone, oestrogens, androgens, paracrine, autocrine
doi: 10.1111/jne.12405
Introduction
Reproduction is tightly regulated by the actions of hormones, both
central and peripheral in origin. The ‘classical’ mechanisms of ster-
oidal control of reproduction have been studied for decades, yet
questions remain about how these hormones interact within the
nervous system to elicit a coordinated response leading to ovula-
tion and fertilisation. The common final pathway to the regulation
of reproductive function is dependent on the appropriate
functioning of the hypothalamic-pituitary-gonadal (HPG) axis. The
proper coordination of the HPG axis relies largely on the inputs
that regulate gonadotrophin-releasing hormone (GnRH) release
from hypothalamic neurones. In recent years, numerous nonclassi-
cal mechanisms have been uncovered, including newly understood
membrane, autocrine and paracrine actions of steroid hormones. In
addition, novel neuropeptides have been added to the list of

neuroendocrine mediators such as the truncated GnRH [GnRH-(1-
5)], as well as the inhibitory gonadotrophin release-inhibitory hor-
mone (GnIH). Together, these recently appreciated events have
changed our understanding of the interaction of the HPG axis and
the relationship between the periphery and the central nervous sys-
tem in the regulation of reproduction.
Control of the LH surge
Central nervous system (CNS) regulation of the LH surge
As reviewed previously, oestradiol membrane signalling, comprising
oestradiol (E
2
) signalling that is initiated at the cell membrane,
plays an important role in the CNS synthesis of progesterone (neu-
roP) needed for oestrogen positive-feedback of the LH surge (1).
Although the preovulatory rise in circulating E
2
is essential for
stimulating gonadotrophin release (2–4), progesterone is also nec-
essary for the LH surge (5–9). In ovariectomised rats and mice, E
2
induces an LH release (10) and LH levels are augmented by addi-
tional application of progesterone (11,12). Blocking progesterone
receptor (PR) or progesterone synthesis prevents the E
2
-induced
GnRH and LH surges in ovariectomised rats (5,13) and arrests the
oestrous cycle in intact female rats (14). Most critically for this dis-
cussion, ablation of PR in kisspeptin (KP)-expressing neurones abro-
gates oestrogen positive-feedback (15), indicating that that both E
2
and progesterone are necessary for surge release of LH.
Where does neuroP act to influence the LH surge? It is well
established that GnRH neurones themselves do not express the req-
uisite steroid hormone receptors, oestrogen receptor (ER)aand PR
(16,17). There is now solid evidence that the LH surge ‘pattern gen-
erator’, which integrates steroid hormone information and regulates
oestrogen positive-feedback is a population of KP-expressing neu-
rones of the rostral periventricular nucleus of the third ventricle
(RP3V), an area that includes the anterior periventricular nucleus
and the anteroventral periventricular nucleus (18–25). Kiss1 neu-
rones in the RP3V are critical for GnRH secretion because KP
released from Kiss1 neurones activates GnRH neurones via GPR54,
a G-protein coupled receptor that binds KP (26–28). Although much
of the work on steroid regulation of KP and its gene, Kiss1, has
focused on E
2
(29,30), it is now evident that E
2
and neuroP func-
tion together to regulate KP. First, both ERaand PR are needed for
positive-feedback of the LH surge (31,32), and both have been loca-
lised in KP neurones, although neither are found in GnRH neurones
(20,33). Consistent with the need for E
2
-induced PRs for the LH
surge, a substantial number of KP neurones in RP3V and the
arcuate nucleus of the hypothalamus (ARH) express PR after E
2
treatment (25,30,33,34). Coincident with this, rising E
2
levels during
pro-oestrus induce neuroP synthesis (14,35).
A combination of in vitro and in vivo experiments have demon-
strated that neuroP acts on KP neurones to mediate oestrogen pos-
itive-feedback (Fig. 1). Integrated steroid signalling was studied in a
cell line (mHypoA51s) that approximates ‘sexually mature’ female
hypothalamic neurones. These immortalised neurones have the
characteristics of post-pubertal RP3V KP neurones because they
express ERa, PR and KP (36). As with KP neurones in vivo,E
2
and
the ERaagonist, PPT, induced KP and PR in mHypoA51s.
Significantly, E
2
-induced PR up-regulation was dependent on an
intracellular ER, whereas KP expression was stimulated by membrane-
impermeable E
2
(E
2
coupled to bovine serum albumin; E-6-BSA). These
data suggest that anterior hypothalamic KP neurones utilise both
membrane-initiated and classical nuclear oestrogen signalling to up-
regulate KP and PR, which are essential for the LH surge.
The nature of progesterone signalling in KP neurones remains to
be clarified. In addition to classical nuclear PR, there are intriguing
suggestions that KP neurones in vitro and in vivo have membrane
progesterone receptors, especially mPRb(37). The mPRs are seven-
transmembrane proteins that activate G proteins that belong to the
progestin and adipoQ receptor (PAQR) family not the classic G pro-
tein-coupled receptor (GPCR) family (38–40). PAQRs can signal
through mitogen-activated protein kinase activation and increasing
[Ca
2+
]
i
(41–47); but see also (48). Studies in mHypoA51s indicate
that classical PR is responsible for progesterone-induced signalling
events. Treatment of E
2
-primed mHypoA51s with progesterone
induces a rapid increase in free cytoplasmic calcium ([Ca
2+
]
i
), which
appears to be responsible for the release of KP induced by
progesterone, whereas inhibition with RU486 prevents the [Ca
2+
]
i
increase (36).
In vivo, preliminary experiments have demonstrated that exoge-
nous progesterone rescued the LH surge in females whose hypothala-
mic steroidogenesis was blocked with the CYP11A1 inhibitor
aminoglutethimide (AGT) (49). In AGT-treated animals, infusions of
progesterone or KP into the diagonal band of Broca induced an LH
surge, confirming that KP operates downstream of neuroP. Finally, KP
knockdown in the RP3V prevented the E
2
-induced LH surge (49). Most
importantly, the ablation of PR in KP neurones in ovariectomised mice
abrogates E
2
positive-feedback (15) demonstrating that that both E
2
and neuroP are necessary for the surge release of LH.
Molecular mechanisms of GnRH-(1-5) action
The decapeptide GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-
NH2) is highly conserved across species, suggesting its functional
importance throughout evolution (50). GnRH is primarily known for
its role in regulating reproductive function and behaviour via inter-
action with KP and its cognate receptor, GPR54, in the hypothala-
mus (51–57). Within each oestrous cycle, a rapid increase in GnRH
secretion culminates in an LH surge, which precedes the onset of
sexual receptivity and ovulation. In addition to its effects on the
secretion of LH, GnRH can autoregulate its own biosynthesis and
secretion via an ultrashort-loop feedback mechanism (58–62).
GnRH not only functions in its full form, but also can signal via
its metabolite, GnRH-(1-5). GnRH-(1-5) is produced by the cleavage
of GnRH by the zinc metalloendopeptidase EC3.4.24.15 (EP24.15) at
the covalent bond linking the fifth and sixth amino acids (63–65)
(Fig. 2). Localisation of EP24.15 supports the involvement of
EP24.15 in the modulation of hypothalamic GnRH neuronal func-
tion (63,66). EP24.15 immunoreactivity is sensitive to hormonal
fluctuations: increasing on pro-oestrous day of the rat oestrous
cycle within the median eminence, with a peak expression coincid-
ing with the LH surge (63). Unlike GnRH, GnRH-(1-5) robustly
©2016 British Society for Neuroendocrinology Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12405
2of12 L. M. Rudolph et al.

stimulates GnRH gene expression (67) and stimulates GnRH secre-
tion (68). Moreover, the GnRH facilitation of lordosis behaviour is
actually mediated by its metabolism to GnRH-(1-5) (69).
Interestingly, studies show that GnRH-(1-5) does not bind to
the GnRH receptor (51) but binds to two orphan GPCRs: GPR101
(70) and GPR173 (71,72) (Fig. 2). Both GPR101 and GPR173 are
members of the Rhodopsin class of receptors. The Rhodopsin fam-
ily is the largest of the five groups of orphan receptors with 672
members of which 63 have no known ligands. Both GnRH-(1-5)-
binding GPCRs are highly conserved and are highly expressed in
the hypothalamus (Allen Brain Bank) (73,74). In several species,
the coding sequence for GPR101 is located on the X chromosome
in a band that is syntenic between species (75). In mouse,
GPR101 mRNA is 2186 bases, encoding a seven-transmembrane
receptor that is approximately 51 kDa (76). The sequenced
GPR173 mRNA is 1122 bases, which translates to a 42-kDa
seven-transmembrane receptor (73). Functional studies suggest
that GnRH-(1-5) retards the cellular migration of neural cells via
GPR173 (71–73). By contrast, GnRH-(1-5) may stimulate cellular
migration and invasion of the extracellular matrix in endometrial
cells via GPR101 (70,77).
These studies support the idea that GnRH-(1-5) represents
another layer of regulatory complexity in tissues where GnRH is
also produced. The identification of an endogenous ligand to an
orphan GPCR is important because these receptors may have thera-
peutic potential (74). Furthermore, the identification of a GPCR that
binds GnRH-(1-5) may help resolve some of the current quandaries
regarding the actions of GnRH (agonist/antagonist) and enhance
our understanding in the evolution of peptide metabolism and
processing.
Role of GnIH in avian reproductive system; regulation of
GnIH by photoperiod and stress and the effects of these
changes on reproductive behaviours
Although GnRH and its metabolite, GnRH-(1-5), are known for pro-
moting reproduction-related functions in the HPG axis, a more
recently discovered hormone has been implicated as a potential
brake on the HPG system. GnIH has received attention because of
its role in the inhibition of activity of components of the HPG axis,
including a reduction of sexual behaviour (78–86). Despite a great
deal of investigation into its specific functions and the factors that
Kiss Neuron
E2ERα
3β-HSD
ERα
E2
Kisspeptin
mRNA/protein
MAPK p
p
Src
PR
+
+
+
Kisspeptin
release
GPR54
GnR
H
E2
ERα
neuroP
PREG
O
[Ca2+]i
LH
P450scc
Gonadotroph
Astrocyte
mGluR1a
Fig. 1. A model showing proposed actions of oestradiol (E
2
) on hypothalamic cells. In kisspeptin (Kiss1) neurones, E
2
acts at both membrane and nuclear
oestrogen receptors. During di-oestrus, classical nuclear E
2
signalling induces progesterone receptor (PR) expression in Kiss1 neurones in the rostral periventric-
ular nucleus of the third ventricle (RP3V). On pro-oestrus, rising E
2
leads to transactivation of mGluR1a in astrocytes, which increases [Ca
2+
]
i
, leading to the
conversion of cholesterol to pregnenolone (PREG) by the P450scc enzyme and the conversion of PREG to progesterone (neuroP) by 3b-hydroxysteroid dehydro-
genase (HSD). Simultaneously, E
2
activates an oestrogen receptor (ER)a-mGluR1a complex in neurones leading to the expression of Kiss1. Newly synthesised
neuroP diffuses out of the astrocytes and activates E
2
-induced PR, which has been trafficked to the Kiss1 neuronal membrane. This leads to a series of events
culminating in Kiss1 secretion onto GPR54 expressing gonadotrophin-releasing hormone (GnRH) neurones. Signalling through PR in Kiss1 neurones induces
Kiss1 release, activating GnRH neurones and triggering the E
2
-induced luteinising hormone (LH) surge from anterior pituitary gonadotrophs. MAPK, mitogen-
activated protein kinase.
©2016 British Society for NeuroendocrinologyJournal of Neuroendocrinology, 2016, 28, 10.1111/jne.12405
Peripheral and central hormonal control of reproduction 3of12

regulate GnIH, the full range of actions of GnIH within the central
nervous system remain unknown. At present, we know that, in
birds, GnIH projects to GnIH receptor-expressing GnRH-I and -II
neurones in addition to the median eminence (84,87). In several
species of mammals, GnIH projects to and also influences the activ-
ity of GnRH neurones (85,88), as well as the external layer of the
median eminence (88–92), although this latter finding remains dis-
puted (85,93). There are GnIH projections to multiple other brain
areas (e.g. brainstem) and possibly to the spinal cord (84,93),
although the function of GnIH in these extra-hypothalamic areas
remains obscure. The GnIH content of the brain is influenced by
changes in day length and the associated changing melatonin sig-
nal in seasonal breeders (84,94–99). In birds, despite the influence
of GnIH on GnRH neurones, it appears that GnIH does not influ-
ence the termination of reproduction at the end of the breeding
season. Rather, it is more likely that GnIH plays a role in temporary
reproductive suppression within the breeding season in response to
different physiological stimuli, such as stress (84,100–102).
The action of GnIH is not restricted to the brain and the anterior
pituitary gland. GnIH and its receptor (GPR147) are synthesised
in the gonads of both sexes of all vertebrates studied to date
(103–108). Furthermore, in birds, GnIH-producing neurones in the
brain project to the pars nervosa, suggesting that GnIH is released
directly into the bloodstream (G. Bentley, unpublished observations).
If confirmed, then not only can locally produced GnIH act within
the gonads, but also neurally produced GnIH could be released to
the general circulation and act upon peripheral targets.
It is possible that GnIH-producing neurones can be subdivided
into heterogenous subpopulations that respond to unique environ-
mental and physiological cues. For example, GnIH neurones express
melatonin receptor (MelR) and glucocorticoid receptor (GR) mRNA.
However, not all of the GnIH neurones express MelR or GR (98,109)
and it is not known whether single GnIH neurones can express
both MelR and GR, suggesting that there could be MelR- and GR-
specific subpopulations of GnIH neurones, each with potentially dis-
tinct functions. Thus, it remains to be determined whether or how
melatonin and glucocorticoids interact to influence GnIH action
within the brain.
In birds and mammals, melatonin and corticosterone can act on
the gonadal GnIH system. This suggests the possibility that the
neural and gonadal GnIH systems could differentially respond to
hormones and, together, could coordinate a response to circulating
hormones (perhaps via direct innervation of the gonad). Unfortu-
nately, only in vitro preparations can be used to answer this ques-
tion. Without separating the gonads from the blood circulation and
from potential neural input, it is impossible to determine gonadal
responses to a changing hormonal environment, especially if GnIH
is present in circulating blood. However, in vivo studies in this area
pGlu
His
Trp
Ser
Tyr Gly
Leu
Arg
Pro
Gly
GnRH
pGlu
His
Ser
Tyr
EP24.15
NH2
GnRH-(1-5)
GPR101
COOH
GPR173
NH2
COOH
Trp
Fig. 2. Gonadotropin-Releasing Hormone (GnRH) peptide processing and action. The decapeptide, GnRH, is processed extracellularly to form the metabolite,
GnRH-(1-5) by the zinc metalloendopeptidase, EC3.4.24.15 (EP24.15; 66, 73). The metabolite, GnRH-(1-5), exerts is biological activities via 2 putative receptors,
the G-protein coupled receptors (GPR) GPR101 and GPR173 (70, 71).
©2016 British Society for Neuroendocrinology Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12405
4of12 L. M. Rudolph et al.

could also be very informative, especially if localised blockade of
GnIH receptor could be induced in the gonads.
GnIH responses to chronic stress have been documented in male
and female rats, with a significant impact on reproduction
(109–111). To date, there has been only one study on chronic stress
effects upon GnIH in birds with sex-specific effects of treatment.
Female European starlings (Sturnus vulgaris) exhibited increased
ovarian GnIH expression compared to their nonstressed counter-
parts and were also reported not to ovulate, whereas nonstressed
animals did (111). Acute stressors can certainly influence the avian
GnIH system, although these effects appear to depend on the spe-
cies, the time of year, the sex of the bird and the stressor
(112–114). In addition, some stressors influence the gonads directly
rather than via neural GnIH (112). The same is true for chronic
housing stress in European starlings, as noted above (111). Thus, it
is clear that neural and GnIH systems can respond differently to
any particular stressor, regardless of whether it is acute or chronic.
Further studies in this area should determine the response of gona-
dal and neural GnIH systems to stressors and hormones, and
should also assess communication between these GnIH systems in
a variety of species.
Local regulation of gonadal function
Autocrine and paracrine regulation of testicular function:
molecular pathways involved in testis pathophysiology
leading to infertility
Gonadotrophins are key regulators of male gonadal function. LH
and follicle-stimulating hormone (FSH) released from the pituitary
reach the testis and exert their effects through receptors located in
the plasma membrane of Leydig and Sertoli cells, respectively
(115,116). In addition, local factors and hormones influence testicu-
lar function via paracrine and autocrine mechanisms. Several mole-
cules that reach the testis and/or are locally produced in the gonad
regulate the activity of different cell types (e.g. Leydig cells, Sertoli
cells, mast cells, macrophages, myofibroblasts), include peptides
(117), neurotransmitters (118), neurohormones (119), cytokines
(120) and prostaglandins (PGs) (121).
In this context, the neurohormones serotonin (122), melatonin
(123) and corticotrophin-releasing hormone (CRH) (124) act in the
testes as important negative regulators of cAMP and androgen pro-
duction. Serotonin, melatonin and CRH can be produced within the
CNS and secreted into peripheral circulation, or locally synthesised
in the testes (125,126). Melatonin and also serotonin inhibit
steroidogenesis via their 5-HT
2
receptor- and Mel1a receptor-
mediated signalling pathways, which influence CRH centrally
(125,127) and in the testes (127,128). This CRH-mediated inhibition
of steroid production occurs through the activation of tyrosine
phosphatases, which reduces the phosphorylation of extracellular
regulated kinase (ERK) and c-Jun N-terminal kinase, and subse-
quently down-regulates c-jun, c-fos and steroid acute regulatory
protein (StAR), thereby inhibiting testosterone production (128).
Melatonin has been postulated to have a physiological role as a
paracrine signalling molecule, directly regulating the production of
factors (e.g. immune, interleukin-2) in its immediate vicinity (129).
Recent observations show that melatonin modulates local cellular
activity in testicular immune cells, inducing the expression of
antioxidant enzymes and reducing the generation of reactive oxy-
gen species in mast cells. In testicular macrophages, melatonin inhi-
bits cell proliferation, the expression of proinflammatory cytokines,
interleukin-1band tumour necrosis factor a, and PG production
(130). PGs are derived from arachidonic acid by the action of indu-
cible isoenzyme cyclooxygenase (COX). In testicular biopsies of men
with impaired spermatogenesis, COX-2 is expressed in immune cells,
highlighting their relevance in testicular inflammation associated
with idiopathic infertility (131). Furthermore, Leydig and Sertoli cells
also produce PGs and express several prostanoid receptors
(132,133), suggesting autocrine/paracrine action in testicular
somatic cells.
PGD2 has a stimulatory effect on basal testosterone production
in Leydig cells (134), whereas PGF2aexerts an inhibitory role in the
expression of the StAR and 17b-hydroxysteroid dehydrogenase
(HSD), as well as in the synthesis of testosterone induced by human
chorionic gonadotrophin (hCG)/LH (133), demonstrating that the
role of PGs on steroidogenesis, spermatogenesis and ultimately fer-
tility depends on the specific PG in question.
Recent research indicates that multiple local signals influence
testicular physiology and are involved in the pathogenesis or main-
tenance of human infertility. Notably, male infertility results from
endocrine dysfunctions associated with the hypothalamic-pituitary-
testicular axis only in a small number of cases (135), suggesting
the source of infertility likely occurs within local, intra-testicular
pathways. Thus, new insights about how cell–cell interactions
within the testes affect testicular function and fertility will con-
tribute to the understanding of male reproductive physiopathology,
and future studies focusing on testicular paracrine and autocrine
interactions may lead to new therapeutic approaches to idiopathic
male infertility.
Follicular development, corpus luteum and progesterone
regulation of ovarian vascularisation and molecular
pathways involved
Similar to testicular functions including spermatogenesis and
steroidogenesis, ovarian follicular development and regression is a
continuous and cyclic process that depends on a number of endo-
crine, paracrine and autocrine signals. In healthy tissues, physiologi-
cal angiogenesis is mainly limited to the reproductive system. The
ovarian vasculature is closely associated with preovulatory follicle
and corpus luteum during the ovarian cycle and is one of the few
sites where nonpathological development and regression of blood
vessels occurs in the adult. Recently, local factors such as vascular
endothelial growth factor A (VEGF-A) and angiopoietins, which act
specifically on vascular endothelial cells or pericytes and smooth
muscle to control angiogenesis or angiolysis, were identified in the
growing follicle and corpus luteum of several species, including
humans (136).
VEGF-A is a key angiogenic factor involved in the formation of
new blood vessels within many tissues. It is required to initiate the
©2016 British Society for NeuroendocrinologyJournal of Neuroendocrinology, 2016, 28, 10.1111/jne.12405
Peripheral and central hormonal control of reproduction 5of12

formation of new immature vessels by promoting endothelial cell
proliferation and vascular permeability. Inhibition of VEGF-A and
angiopoietin 1 (ANGPT1) action in rat ovaries by intrabursal admin-
istration of VEGF-A-Trap or ANGPT1 antibodies, respectively, pro-
duces an imbalance in the ratio of anti-apoptotic : pro-apoptotic
proteins leading to greater follicular atresia (137,138). In addition,
VEGF-A prevents apoptosis and stimulates the proliferation of gran-
ulosa and theca cells of antral follicles through a direct interaction
with its KDR receptor localised in granulosa cells, a pathway that
involves phosphoinositide 3-kinase (PI3K)/AKT (139). Furthermore,
in vitro studies performed in early antral follicles and granulosa cell
cultures isolated from rat demonstrate that VEGF acts directly on
follicular cells synergistically with FSH and E
2
, preventing apoptosis
and stimulating proliferation, thus promoting follicular development
and the selection of the follicle to ovulate (140). Such work
reported a direct role for VEGF in early antral follicles mediated by
the PI3K/AKT and ERK1/2 pathways, besides the classical and well
known proangiogenic function. Together, these data support the
notion that angiogenic factors have an important role in controlling
ovarian function.
In vitro studies have shown that Notch signalling is critical for
the survival of luteal cells isolated from pregnant rats (141). Local
Notch inhibition decreases progesterone levels and cell survival,
confirming that Notch has a direct action on both steroidogenesis
and luteal viability (141). The Notch signalling pathway is a cell–cell
communication pathway that is evolutionarily conserved from Dro-
sophila to humans. To date, four different Notch receptors (Notch1,
2, 3 and 4) and five different ligands (Jagged-1 and -2 and DLL-1 -
3 and -4) have been identified in mammals. This Notch system reg-
ulates cell fate, proliferation and death. The Notch genes encode
transmembrane receptors, which, upon binding their ligand, are
cleaved, releasing the intracellular domain. The intracellular portion
of the receptor translocates to the nucleus to act as a transcrip-
tional coactivator, regulating cell fate genes (142).
Moreover, in the rat, there is an interaction between the Notch
signalling pathway and progesterone that maintains the functional-
ity of the corpus luteum (143). Notch signalling augments P450scc
synthesis, leading to an increased synthesis of progesterone, which
in turn regulates the activated intracellular Notch domain. Thus,
Notch induces progesterone production in vitro through the activa-
tion of cytochrome P450 cholesterol side chain cleavage enzyme
(P450scc) and decreases apoptosis-mediated cell death. This is the
first evidence that there is cross-talk between the Notch signalling
system and progesterone, which increases the survival of luteal
cells. Also, the Notch/PI3K/AKT signalling pathway might be inter-
acting with progesterone, intensifying the survival role of this hor-
mone in luteal cells. Nevertheless, future studies are required to
thoroughly investigate this newly discovered Notch-progesterone
relationship and how it contributes to ovarian function and repro-
duction as a whole.
Ovarian kisspeptin and its role in follicular development
Reproduction in females requires an LH surge, which is centrally
regulated by KP. However, KP is found in many peripheral organs
(144,145), in particular, the ovary, which expresses KP and its
receptor, GPR54, suggesting a role for KP in the peripheral control
of reproductive events. KP expression in the ovary fluctuates
throughout the oestrous cycle, strongly suggesting that it may be
involved locally in the ovulatory cycle and luteinisation (146–148);
but see also (28). However, the mechanisms of action of KP in the
ovary, such as paracrine or autocrine functions remain largely
unknown.
A recent study demonstrated that intraovarian administration of
a KP antagonist (p234) delays vaginal opening and alters the oes-
trous cycle in rats (147). Additionally, local administration of exoge-
nous KP decreases antral follicle and corpora lutea number in
fertile and subfertile rats, which was reversed by p234 treatment,
suggesting that KP also participates in both follicular development
and ovulation at the level of the ovary (149). Moreover, during ovu-
lation in humans and nonhuman primates, ovarian KP and GPR54
mRNA increases with other ovulation-associated genes, such as
COX-2 and progesterone receptor. The ovarian administration of the
COX-2 inhibitor, indomethacin, disrupted the ovulatory process in
rats, supporting the idea of a local role of KP and GRP54 in ovula-
tion (150). It appears that KP regulates progesterone secretion from
luteal cells as well. In isolated chicken granulosa cells, KP stimulates
progesterone secretion, possibly by directly altering levels of
steroidogenic enzymes, including StAR, P450scc, which converts
cholesterol to pregnenolone, and 3b-HSD (151), which converts
pregnenolone to progesterone. Similarly, in rat luteal cells, KP
increased progesterone production via ERK1/2 signalling and
increased the expression of StAR and CYP11A mRNA (152). Further-
more, administration of a GPR54 antagonist, p234, inhibited pro-
gesterone secretion in granulosa cell cultures treated with hCG,
implicating KP in the luteinisation of granulosa cells (148). Together,
these data suggest a potential role of KP in the local control of
ovarian function, potentially via progesterone synthesis. These and
future studies involving paracrine and autocrine actions of ovarian
KP will clarify the molecular mechanisms involved in the regulation
of follicular development and ovulation during reproductive life and
ovarian ageing.
Although a decreased follicular pool indicates physiological age-
ing of the ovary (153), an increased rate of follicular loss is also a
pathology that affects the follicular reserve pool, and thereby fertil-
ity, in humans and other mammals (154). Reproductive ageing in
women begins with shortened menstrual cycles, smaller increases
in FSH and decreased levels of inhibin (155), which results in accel-
erated follicular growth and premature exhaustion of the follicular
pool. One of the mechanisms involved in ovarian ageing is
increased sympathetic nerve activity. Ovaries of postmenopausal
women (≥51 years old) have a higher density of innervation com-
pared to age-matched controls (156,157). In the rat, reproductive
ageing is associated with increased ovarian sympathetic activity,
which is strongly correlated with the spontaneous appearance of
follicular cysts and a loss of preantral follicles (158,159). Indeed,
the highest sympathetic innervation is found in postmenopausal
women, suggesting a correlation between ageing-induced infertility
and sympathetic nerve activation. Recent findings indicate that
sympathetic innervation may be controlling age-induced infertility
©2016 British Society for Neuroendocrinology Journal of Neuroendocrinology, 2016, 28, 10.1111/jne.12405
6of12 L. M. Rudolph et al.

via regulation of KP because ovarian sympathectomy diminishes KP
levels (A. Paredes, unpublished observations). Additionally, during
reproductive ageing, KP expression in the ovary increases from the
subfertile to infertile period and is directly correlated with the
increase in ovarian norepinephrine observed with ageing (149,158),
suggesting that KP may be directly controlled by sympathetic inner-
vation of the ovary (147), as well as supporting the idea that KP is
regulated by the adrenergic system and that both the adrenergic
system and KP participate in the local regulation of follicle develop-
ment and ovulation during reproductive ageing. Furthermore, KP is
involved in follicular dynamics: intraovarian administration of KP
produced an increase in the numbers of corpora lutea and type III
follicles in fertile and subfertile periods, which was reversed by KP
receptor antagonism. Future studies should address the potential
autocrine and paracrine roles of KP in the ovary, specifically the
interaction of KP, steroidogenic pathways and sympathetic innerva-
tion and how they relate to reproductive outcomes across the
lifespan.
Morphological changes in ARH initiated by oestradiol
membrane signalling that mediate lordosis behaviour
Another key component to reproduction in rodents is female sexual
receptivity, which is mediated by E
2
-dependent alterations in
hypothalamic neuronal structure. Although the molecular bases of
E
2
-dependent facilitation of female sexual receptivity have more
recently been described in detail, the understanding that steroid
hormones exert behavioural effects via changes in neural morphol-
ogy is a well established phenomenon. The most well known exam-
ple of E
2
-induced changes in dendritic structure regulating
memory-related behaviour is from the hippocampus (160), whereas
E
2
-induced changes in dendrites in the hypothalamus have also
been known for some time (161). Indeed, changes in dendritic mor-
phology are critical for the lordosis-regulating circuit (162), which
extends from the ARH to the medial preoptic nucleus (MPN), to the
ventromedial nucleus of the hypothalamus (VMH). Recent studies
have begun to clarify the molecular mechanisms by which morpho-
logical changes in the ARH-MPN-VMH circuit allow for expression
of lordosis behaviour. The primary step of E
2
signalling in the ARH
occurs via ERatransactivation of mGluR1a, which initiates morpho-
logical changes that are coincident with and required for the dis-
play of lordosis behaviour. Within 4 h after E
2
treatment, immature,
filapodia-like dendritic spines are formed in the ARH (162). Twenty-
four hours after E
2
treatment, there is a shift in the proportion of
dendritic spines, with a decrease in filapodia and a concomitant
increase in mature, mushroom-shaped spines (162). The formation
of new spines is necessary for the E
2
-induced lordosis because
blocking spine formation significantly reduces the expression of
sexual receptivity (162).
Although it appears that spinogenesis is initiated by the action
of E
2
at membrane ERa, it is unclear what molecular mechanisms
underlie spine maturation. Evidence from other circuits suggests a
role for the G-protein coupled ER, GPR30, in spine maturation and
stabilisation. GPR30 is localised in spine heads, associates with
PSD-95, and is regulated by E
2
(163,164). In the dorsal
hippocampus, the GPR30 agonist, G1, increases PSD-95 immunore-
activity, suggesting a role for GPR30 in spine maturation (164).
Indeed, this receptor has been implicated in the initiation of lordo-
sis behaviour on the basis that the partial GPR30 agonist but ERa
antagonist, ICI 182,780, facilitates lordosis in E
2
-primed nonrecep-
tive rats (165). Other studies suggest there could be a role for the
STX-activated G
q
-coupled membrane ER in the ARH-MPN circuit
mediating sexual receptivity. STX is a tamoxifen analogue that does
not bind to classical ERaor GPR30 but is blocked by the ER antag-
onist ICI 182,780 and has pharmacological profile similar to those
of the ERa-specific agonist, PPT (166–168). STX treatment induces
l-opioid receptor (MOR) internalisation in the MPN and facilitates
lordosis behaviour (169). Alternatively, spine maturation could be
mediated by extra-neuronal mechanisms, such as astrocytic contact
with neurones, which alters dendritic spine formation and stabilisa-
tion (170).
Additionally, it is unclear whether E
2
induces spinogenesis in the
same population of neurones in the ARH that express ERa, the
neuropeptide Y (NPY) neurones, which are the initial site of the
action of E
2
in the ARH-MPN-VMH circuit, or whether E
2
is acting
transsynaptically to induce spines on pro-opiomelanocortin (POMC)
neurones, which release b-endorphin onto MORs in the MPN.
Recent data suggest that the NPY neurones and not POMC neu-
rones undergo spinogenesis, suggesting that spine formation occurs
directly within the neurones where initial E
2
activation of ERa
occurs (171). Regardless of the site of spinogenesis within the ARH,
it is clear that spine maturation in this nucleus is coincident with
lordosis behaviour, and also that blocking spinogenesis here reduces
female sexual receptivity. To a first approximation, the timeline
from E
2
treatment to the presence of mature dendritic spines is
known. However, the time when fully functional synapses appear
remains to be determined. Within 1 h of E
2
treatment, cofilin is
deactivated via phosphorylation, which permits spinogenesis (162),
and, in the MPN, MOR is activated/internalised, indicating that the
ARH to MPN part of the circuit is functional (172). At 4 h post-E
2
treatment, filapodial spines are present, although these thin, labile
spines are not considered to mediate functional synapses (173). At
20 h after E
2
treatment, the first time point when lordosis beha-
viour can be elicited with supplemental hormone treatment, there
is an increase in the proportion of mushroom spines that are gen-
erally assumed to be indicative of functional synapses (162) and
that contain the machinery required for synaptic transmission (e.g.
PSD-95). Future studies should address the time course of this E
2
-
dependent spine maturation and the potential involvement of non-
traditional ER in this process.
Conclusions
Taken together, these recent findings highlight both the redundancy
and complexity of the hormonal control of reproduction: what was
once considered to be a simple, direct circuit with a handful of
steroid hormones and cognate receptors is continually updated with
novel hormone regulators and mechanisms of hormone synthesis
and action. However, the classical aspects of gonadal hormone con-
trol of reproduction remain intact, demonstrating that there are
©2016 British Society for NeuroendocrinologyJournal of Neuroendocrinology, 2016, 28, 10.1111/jne.12405
Peripheral and central hormonal control of reproduction 7of12

multiple levels of control of the HPG axis, both centrally and
peripherally. Future studies will likely only add to this increasingly
complex circuit that regulates reproduction.
Disclaimer
The opinions or assertions contained herein are the private ones of
the authors and are not to be construed as official or reflecting the
views of the Department of Defense or the Uniformed Services
University of the Health Sciences.
Received 14 January 2016,
revised 25 May 2016,
accepted 20 June 2016
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