Role of melatonin on embryo viability in sheep

Article (PDF Available)inReproduction Fertility and Development 31(1):82-89 · December 2018with 378 Reads
DOI: 10.1071/RD18308
Abstract
Melatonin is a natural hormone synthesised in the pineal gland, the activity of which is regulated by day-night perception and dictates seasonal rhythms in reproduction in ovine species. Exogenous melatonin, administered via subcutaneous implants, is used to prolong the breeding season of ewes and can increase the proportion of pregnant ewes (fertility rate) and litter size. The increased proportion of ewes that become pregnant and the number of lambs born per lambing among melatonin-treated sheep may be caused by increased embryo survival, through enhanced luteal function, reduced antiluteolytic mechanisms, or improved embryo quality. This review focuses on the effects of melatonin on embryo viability and summarises the processes by which this hormone affects the ovary, follicle, oocyte, corpus luteum and embryo. Moreover, the effects of melatonin on the mechanisms of in vivo maternal recognition of pregnancy in sheep and the protective action that it appears to have on the in vitro procedures that are used to obtain healthy embryos are reviewed.
Role of melatonin on embryo viability in sheep
Jose
´-Alfonso Abecia
A
,
C
,Fernando Forcada
A
,Marı´a-Isabel Va
´zquez
B
,
Teresa Muin
˜o-Blanco
A
,Jose
´A. Cebria
´n-Pe
´rez
A
,Rosaura Pe
´rez-Pe
A
and
Adriana Casao
A
A
Instituto Universitario de Investigacio
´n en Ciencias Ambientales de Arago
´n (IUCA), Universidad
de Zaragoza, Facultad de Veterinaria, Miguel Servet, 177, 50013 Zaragoza, Spain.
B
Departamento de Reproduccio
´n Animal, Facultad de Agronomı´a y Veterinaria, Universidad
Nacional de Rı´o Cuarto, Ruta Nacional 36, Km 601, 5800 Rı´o Cuarto, Co
´rdoba, Argentina.
C
Corresponding author. Email: alf@unizar.es
Abstract. Melatonin is a natural hormone synthesised in the pineal gland, the activity of which is regulated by day–night
perception and dictates seasonal rhythms in reproduction in ovine species. Exogenous melatonin, administered via
subcutaneous implants, is used to prolong the breeding season of ewes and can increase the proportion of pregnant ewes
(fertility rate) and litter size. The increased proportion of ewes that become pregnant and the number of lambs born per
lambing among melatonin-treated sheep may be caused by increased embryo survival, through enhanced luteal function,
reduced antiluteolytic mechanisms, or improved embryo quality. This review focuses on the effects of melatonin on
embryo viability and summarises the processes by which this hormone affects the ovary, follicle, oocyte, corpus luteum
and embryo. Moreover, the effects of melatonin on the mechanisms of in vivo maternal recognition of pregnancy in sheep
and the protective action that it appears to have on the in vitro procedures that are used to obtain healthy embryos are
reviewed.
Additional keywords: follicle, ovary, oxidative stress, uterus.
Published online 3 December 2018
Introduction
One of the most remarkable characteristics of ovine reproduc-
tion is its seasonality, which is mediated by photoperiod (Yeates
1949). The main reason why sheep have seasonal reproduction
is to ensure that births occur at the optimal time of the year,
usually spring, which allows the newborn to grow under
favourable conditions of temperature and food availability in
advance of winter (Ortavant et al. 1985). Typically, the breeding
season begins in late summer or early autumn and ends in late
winter or early spring, so that domesticated small ruminants
have retained most of the physiological expressions of repro-
ductive seasonality (Lincoln 1990).
Melatonin, a natural hormone discovered by Lerner et al.
(1958), conveys photoperiod signals to the reproductive neuro-
endocrine axis (Bittman et al. 1983) and is present in all
mammals. Melatonin is synthesised from tryptophan and sero-
tonin in the pineal gland by enzymes whose activities are
regulated by day–night perception. Thus, the pineal gland has
an essential role in the reproductive responses of sheep to
stimulatory and inhibitory photoperiods, because day length
regulates the potency of the negative feedback of oestradiol on
LH secretion, this feedback being responsible for the seasonal
rhythms in reproduction in the ewe (Bittman and Karsch 1984).
Although the use of melatonin in the form of subcutaneous
implants (18 mg melatonin; Melovine/Regulin; CEVA Sante´
Animale) was first used to advance the breeding season in
highly seasonal (Suffolk and Mule cross: English et al. 1986;
Haresign et al. 1990;Wheaton et al. 1990) and Mediterranean
(Rasa Aragonesa: Forcada et al. 1995; Caussenarde du Lot,
Limousine, Tarasconnaise, Rouge de l’Ouest and Lacaune:
Chemineau et al. 1996) sheep breeds, such treatment can also
increase the fertility rate and litter size (Abecia et al. 2011;
Palacı´n et al. 2011). As reviewed below, the increased propor-
tion of ewes that become pregnant and the number of lambs
born per lambing among melatonin-treated sheep may be due
to increased embryo survival, either through improved luteal
function, reduced antiluteolytic mechanisms, or enhanced
embryo quality. Several international groups involved in
multiple ovulation and embryo transfer (MOET) programs in
sheep, goats, cattle and pigs have treated donor ewes with
melatonin implants (Forcada et al. 2006;Buffoni et al. 2014)
or included melatonin in the culture media to improve the
quantity and quality of oocytes and/or embryos in sheep
(Casao et al. 2009;Zhang et al. 2013), goats (Soto-Heras
et al. 2018), cattle (Papis et al. 2007) and pigs (Rodriguez-
Osorio et al. 2007).
CSIRO PUBLISHING
Reproduction, Fertility and Development, 2019, 31, 82–92
https://doi.org/10.1071/RD18308
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The role of melatonin in embryo growth and development
(Abecia et al.2008), as well as in the production and preservation of
mammalian gametes and embryos (Cruz et al. 2014), has been
reviewed elsewhere. In this review, we focus on the effects of
melatonin on embryo viability, particularly in sheep, in vivo and
in vitro, describing the process by which melatonin affects the
ovary, follicle, oocyte, corpus luteum (CL) and embryo, and its role
in the mechanism of maternal recognition of pregnancy in sheep.
Melatonin receptors in organs
In mammals, at least two high-affinity G-protein-coupled
receptors, namely melatonin MT
1
and MT
2
receptors, must be
activated in numerous physiological mechanisms in which
melatonin is involved (for a review, see Dubocovich and
Markowska 2005). MT
1
receptors modulate neuronal firing
(when a neuron emits an action potential or a nerve impulse),
arterial vasoconstriction, cell proliferation in cancer cells and
reproductive and metabolic functions, whereas MT
2
receptors
shift the circadian rhythms in neuronal firing in the supra-
chiasmatic nucleus, inhibit dopamine release in the retina,
induce vasodilation and inhibit leucocyte rolling in arterial beds
(the adhesion of leucocytes to the vascular endothelium during
the inflammatory process) and enhance immune responses
(Dubocovich and Markowska 2005). Those melatonin receptors
are expressed in sheep (Reppert et al. 1994;Coge et al. 2009), in
which expression (or not) of an MT
1
receptor gene polymor-
phism is associated with out-of-season breeding and fertility in
Mediterranean (Sarda (Carcangiu et al. 2009) and Rasa Arago-
nesa (Martı´nez-Royo et al. 2012)) and other (Dorset: Mateescu
et al. 2009) sheep breeds. In addition, we have provided evidence
that MT
1
and MT
2
receptors are present in ram spermatozoa
(Casao et al. 2012). In the female gamete, MT
1
and MT
2
receptors
are expressed in sheep oocytes, cumulus cells and granulosa cells
(Tian et al. 2017), and our group was the first to report the
presence of MT
1
receptors in sheep blastocysts (Casao et al.
2010b). In that study, hatched blastocysts obtained in vitro were
subjected to indirect immunofluorescence with an anti-melatonin
MT
1
receptor antibody (Casao et al. 2010b). Direct observation
by fluorescence microscopy of embryos incubated with the
anti-MT
1
receptor antibody showed intense fluorescence that
corresponded to the perinuclear area of the plasma membrane of
trophoblast cells. Subsequent research with antibodies specific
against each receptor type confirmed the presence of the MT
1
receptor in the perinuclear area, and MT
2
throughout the nucleus.
The gene expression of the two receptors in sheep blastocyst was
verified by quantitative polymerase chain reaction (PCR) anal-
yses (Casao et al. 2018). Those results confirmed the presence of
melatonin receptors in sheep in early embryo development and
have been confirmed in bovine embryos (Wang et al. 2014), as
well as in cumulus and granulosa cells in pigs, but not pig oocytes
(Kang et al. 2009).
Effects of melatonin on in vivo embryo production
Melatonin treatment of donor and recipient ewes in
MOET programs
MOET technology and the inherent laboratory assisted repro-
ductive techniques (ARTs), namely IVM, IVF and in vitro
culture (IVC), were developed for sheep to accelerate genetic
improvement by increasing the number of offspring produced
by superior females, although breeding seasonality precludes
the additional benefits of the technology. In anoestrous sheep,
conception rates decrease after superovulation (Smith et al.
1988). Therefore, embryo production in vivo (from donor
ewes) is performed primarily in the reproductive season of the
ewe, which limits the number of animals that an embryo
transfer team can manage each year. Extending the application
of MOET techniques to ewes in the non-breeding season would
increase labour efficiency and enable the sheep industry to
benefit more from the technology. McEvoy et al. (1998) made
the first attempt to improve embryo production in super-
ovulated ewes in the anoestrous season, but they did not find
differences in the number and quality of embryos from
melatonin-treated compared with untreated donor Border
Leicester Scottish Blackface ewes in anoestrus. Similar
results were reported by Buffoni et al. (2014),whousedmel-
atonintotrytoimproveembryoproductioninMOETsheep
programs in the Dohne Merino breed at 438S, but found no
effects on ovulation, fertilisation or viability rates, nor on the
number of viable embryos per ewe; however, the number of
degenerated embryos in the anoestrous season was highest in
the melatonin-treated group, possibly as a consequence of
seasonal shifts in LH secretion and/or associated effects on
follicle function (Mitchell et al. 2002).
Our group took another approach: evaluating the possibility
of obtaining embryos of high genetic value from select ewes at
the end of their reproductive lives (Forcada et al. 2006). In
senescence, the secretion of melatonin is reduced (Reiter 1992).
In sheep, exogenous melatonin increases LH levels after
gonadotrophin-releasing hormone (GnRH) injections in aged
ewes, which indicates that melatonin can restore the functioning
of the neuroendocrine system in ewes that had been diminished
by senescence (Forcada et al. 2007). In fact, the administration
of melatonin to aged female rats increases GnRH synthesis
(Li et al. 1997) and pituitary responsiveness to GnRH (Diaz
et al. 2000) to levels exhibited by young, cyclic animals. We
used mature Rasa Aragonesa ewes (mean age 10 years) that had
more than eight lambings in their life and were selected for
prolificacy (mean litter size .1.4 lambs per lambing). For two
consecutive years, half the donor ewes received a subcutaneous
melatonin implant, and two common MOET procedures were
performed 40 days (Recovery 1) and 120 days (Recovery 2) after
melatonin implantation. Ewes were slaughtered after Recovery
2 and uterine samples were collected to measure the endometrial
progesterone and oestradiol receptor population. In that study,
exogenous melatonin improved the viability of embryos col-
lected from high-prolificacy aged Rasa Aragonesa ewes after
superovulation. However, the effect of melatonin occurred in
the medium term (i.e. 3 months after implantation), increasing
the proportion of blastocysts and the viability and freezability of
embryos (Forcada et al. 2006).
In a study by Zhang et al. (2013), in addition to the
commercial dose provided by the melatonin implants (18 mg),
other doses (0, 40 or 80 mg per animal) were implanted
subcutaneously into donor and recipient ewes before superovu-
lation and oestrus synchronisation. In that study, both ovulation
Melatonin and embryo viability in sheep Reproduction, Fertility and Development 83
rate (13.4 and 15.1 vs 8.8 CL per ewe for 40 and 80 mg melatonin
vs control respectively; P,0.05) and the number of embryos
recovered from ewes (10.3 and 10.9 vs 6.2 embryos per ewe for
40 and 80 mg melatonin vs control respectively; P,0.05) were
significantly higher in the melatonin-treated than control group
(Zhang et al. 2013). Furthermore, pregnancy and birth rates
were markedly improved for embryos transferred from the 40-
and 80-mg melatonin-treated donors compared with control
ewes, and the melatonin implant in recipient ewes increased
the number of lambs born per embryo transferred.
In humans, women on IVF programs who had sleep disorders
that were treated with melatonin had improved oocyte and
embryo quality, although the sleeping disorder was not allevi-
ated (Eryilmaz et al. 2011).
Thus, it can be concluded that the treatment of donor and
recipient ewes with exogenous melatonin in MOET programs
could be a useful tool to improve the performance of these
procedures.
Interaction between exogenous melatonin treatment and
nutrition
Undernutrition increases embryo mortality and reduces the
pregnancy rates in sheep (for a review, see Forcada and Abecia
2006), primarily because of inadequate oocyte quality or early
embryo development (Abecia et al. 2006). In a series of
experiments by our group that were designed to quantify the
interaction between melatonin treatment and undernutrition at
the embryo level in sheep, some ewes received a melatonin
implant and, 45 days later, all ewes were offered one of two
diets: low (0.5 maintenance requirements) or control
(1.5 maintenance requirements), in the breeding and anoes-
trous seasons (Va´zquez et al. 2009,2010a). Ewes were mated,
their embryos collected and the ovaries used for IVF. Mela-
tonin implants improved embryo quality in anoestrus, partic-
ularly in undernourished postpartum ewes, which exhibited a
significant increase in the number of viable embryos per CL
(0.62 vs 0.23 in the melatonin and control groups respectively;
P,0.01) and in viability rate (83.9% vs 46.6% in the mela-
tonin and control groups respectively; P,0.05; Va´zquez et al.
2010b). However, neither nutrition nor exogenous melatonin
treatment significantly affected the competence of oocytes
during IVF (Va´zquez et al. 2009,2010a). Therefore, melatonin
did not appear to act on oocyte competence. However, oocyte
competence in ewes was affected by season, and melatonin
implants seemed to improve developmental competence in the
seasonal anoestrous period, particularly in experimentally
undernourished ewes (Va´zquez et al. 2010b). Similarly, mel-
atonin implants at lambing in the breeding season improved the
viability of embryos that were recovered from undernourished
ewes, although the effect did not appear to act at the level of
oocyte competence (Va´zquez et al. 2013). These results sug-
gest that the mechanisms underlying the interaction between
exogenous melatonin and the level of nutrition on embryo
development are seasonally regulated. Melatonin may be
useful for reversing or alleviating the adverse effects of
undernutrition on embryo survival in sheep, although the
nutrition–melatonin interactions and their mechanisms require
further investigation.
Effects of melatonin on the ovary, follicle and CL
Effects on the ovary
To identify the optimal conditions for the long-term preservation
of ovaries, Peris-Frau et al. (2017) investigated the effect of
adding melatonin to delay the oocyte aging process during ovary
transport. The addition of melatonin to the collecting media
reduced reactive oxygen species (ROS) levels in mature oocytes
and increased the proportion of blastocysts. Goodarzi et al.
(2018) added various concentrations of melatonin (0, 500, 600,
700 or 800 mM ¼control, M1, M2, M3 and M4 groups respec-
tively) as an antioxidant to sheep ovary preservation medium that
was maintained at either 48Cor208C for 24 h and then quantified
the effects on in vitro embryo production parameters. Melatonin
reduced the decline in fertilisation rate (an indicator of a suc-
cessful IVM), and melatonin supplementation increased the total
cell number of blastocysts (an indicator of embryo quality), with
a mean number of cells in blastomeres at 48C of 86.0, 98.5, 111.5,
125.5 and 126.5 for the control, M1, M2, M3 and M4 groups
respectively (Goodarzi et al. 2018). These experiments showed
that the addition of melatonin to the ovary storage medium had
beneficial effects on sheep oocyte development and embryo
quality by reducing the oxidative stress caused by ROS and
preventing the deterioration of oocytes.
Effects on the follicle
Melatonin is present in the follicular fluid of human (Brzezinski
et al. 1987), porcine (Shi et al. 2009), cattle (Tian et al. 2014)
and juvenile goat (Soto-Heras et al. 2018) follicles. The physi-
ological role of melatonin in follicular fluid is not fully under-
stood but, as Tamura et al. (2012) suggested, melatonin may be
the most effective antioxidant in the follicle because ROS are
produced within the follicle, especially during ovulation. Fur-
thermore, excessive ROS cause oxidative stress and can damage
oocyte and granulosa cells. In addition, the presence of mela-
tonin receptors in granulosa cells suggests that melatonin may
be involved in in vivo oocyte maturation, because granulosa
cells are the only somatic cells that closely interact with the
oocyte from the moment the follicle forms until the release of the
oocyte at ovulation (for a review, see Tamura et al. 2009).
Apparently, melatonin modulates steroidogenic gene expres-
sion in the ovary (Maganhin et al. 2014;Lima et al. 2015) and
regulates the response to LH and luteinisation (He et al. 2016)
via the MT
1
receptor.
To assess follicular development and oocyte quality in
melatonin-treated and untreated anoestrous Chios ewes, Tsili-
gianni et al. (2009) used laparoscopic observation and IVF in
two successive ovum pick-up (OPU) procedures. Oocytes col-
lected from the second OPU underwent IVM, IVF and IVC. The
number of large follicles was highest among the melatonin-
treated ewes in the first OPU and tended to be so in the second; in
addition, more of the oocytes collected from melatonin-treated
animals were fertilised and developed in vitro than from
untreated animals. It was concluded that melatonin is a potent
regulator of follicle development and oocyte competence during
the anoestrous period in the ewe.
Using a goat model, Berlinguer et al. (2009) were the first to
demonstrate that melatonin treatment affected follicle growth
84 Reproduction, Fertility and Development J.-A. Abecia et al.
and development by modifying the wave growth pattern by
increasing the turnover of dominant follicles (melatonin caused
more follicular waves because the waves were shortened by
around 2 days compared with waves in untreated control goats).
Furthermore, melatonin increased the rate of cleaved oocytes
compared with control animals (82.5% vs 63.4%; P,0.001),
and advanced the timing of embryo development and enhanced
the blastocyst rate (31.5% vs 16.3%; P,0.01).
Effects of melatonin on the CL and progesterone secretion
Melatonin has a direct effect on the CL by increasing proges-
terone (P4) production: a melatonin perfusion cannula system
in vivo and the culture with melatonin of luteinised granulosa
cells in vitro significantly stimulated P4 secretion for 30–60 min
in vivo and by granulosa cells in vitro after two days of culture
(Durotoye et al. 1997). In addition, melatonin significantly
increased P4 production in granulosa cells in vitro after 2 days in
culture, demonstrating that melatonin can act directly on the CL
to increase P4 production; this could explain the improved luteal
function that occurs late in the breeding season after prolonged
exposure of ewes to melatonin, when, at ram introduction, both
cyclic and non-cyclic melatonin-treated ewes that responded by
ovulating had higher mean plasma P4 concentrations than did
untreated ewes (Abecia et al. 2006).
In another experiment (Abecia et al. 2002), ewes (n¼5)
were subjected to an intravenous melatonin challenge (3 mg/(kg
bw)
0.75
). After the challenge, all ewes on Day 7 and three ewes
on Day 10 exhibited a P4 response to melatonin, which was
defined as an increase in plasma P4 concentrations in at least two
consecutive samples collected in the post-treatment period that
were above the mean þ2SD of the values in the pretreatment
period. In addition, a significant effect of melatonin was
observed on overall plasma P4 concentrations compared with
before the challenge on both Day 7 (0.61 vs 0.73 ng mL
1
before
and after the challenge respectively; P,0.01) and Day 10 (1.16
vs 1.30 ng mL
1
before and after the challenge respectively;
P,0.05). Additional evidence of the effect of melatonin on P4
secretion has been reported by Manca et al. (2014), who found
that the mean area of the CL and plasma P4 concentrations in
sham-operated ewes were significantly higher than in pine-
alectomised ewes. Collectively, the evidence suggests that
melatonin affects the growth pattern of follicles and the ste-
roidogenic capacity of the CL.
Effects on the uterus and oviducts
Several studies have focused on the effects of melatonin on the
uterus, specifically on the uterine receptor population, prosta-
glandin (PG) F
2a
secretion or uteroplacental haemodynamics. In
fact, using a mid- to late gestation ovine model of intrauterine
growth restriction, uteroplacental blood flow and fetal growth
were examined during supplementation with 5 mg day
1
dietary
melatonin (Lemley and Vonnahme 2017). Maternal nutrient
restriction decreased uterine arterial blood flow, whereas mel-
atonin supplementation increased umbilical arterial blood flow
compared with non-supplemented controls. Conversely,
Gimeno et al. (1980) reported that melatonin blocks in vitro
generation of PGF
2a
by the uterus, and that melatonin affects the
neurosecretory function of the hypothalamo–neurohypophysial
complex, possibly via mechanisms involving cholinergic
transmission and/or PG biosynthesis (Bojanowska and Forsling
1997). The addition of melatonin to the culture medium of ovine
endometrium collected on Day 15 after mating reduced the
secretion of PGF
2a
, but only in the case of ewes fed a low-energy
diet, and these ewes exhibited the highest embryonic mortality
(Abecia et al. 1999). Melatonin-treated ewes during anoestrus
had higher plasma P4 concentrations during the male effect-
induced luteal phase than did untreated ewes (Abecia et al.
2006), which may reflect stronger downregulation of the pro-
gesterone receptor by P4 in the melatonin-treated group. Given
that steroid-modulated melatonin receptors are present in the rat
uterus (Zhao et al. 2000,2002), the effects of melatonin on
endometrial physiology may be mediated through changes in the
steroid receptors of the uterus. Our group first reported changes
in the endometrial sensitivity to steroids in undernourished ewes
caused by exogenous melatonin (Va´zquez et al. 2013); specif-
ically, melatonin increased the expression of the progesterone
receptor in the deep glandular epithelium and decreased oes-
trogen ERareceptor expression in the deep stroma of under-
nourished, melatonin-implanted ewes. However, neither
melatonin nor nutrition treatment affected ERaand progester-
one receptor expression in the endometrium during anoestrus.
Furthermore, in undernourished ewes, melatonin implants were
associated with increased embryo viability during anoestrus
(Va´zquez et al. 2013). Overall, the uterine expression of PGR
mRNA was reduced by melatonin treatment (P,0.01), and the
interaction effect was not significant in the reproductive season.
In control and undernourished ewes, melatonin reduced pro-
gesterone receptor expression in the reproductive season.
However, in anoestrus, none of the variables studied was
affected by nutrition, melatonin or their interaction (M. I.
Va´zquez, C. Sosa, A. Meikle, F. Forcada and J. A. Abecia,
unpubl. data). The expression of ERatended to be greater in the
reproductive than it was in the anoestrous season. Undernutri-
tion tended (P¼0.09) to increase the expression of ERa(ESR1)
mRNA, but the effect was observed in the melatonin-treated
ewes in the reproductive season only. Season, nutrition, mela-
tonin and their interactions did not have a significant effect on
insulin-like growth factor (IGF) 1 expression. However, in the
oviducts, expression of IGF2 and IGF1 receptor (IGF1R) was
affected by melatonin and its interaction with nutrition
(P,0.01); exogenous melatonin had no effect on PGR and
ESR1 mRNA expression, but it did increase IGF1 and IGF2,
most strongly in undernourished ewes (M. I. Va´zquez, C. Sosa,
M. Carriquiry, F. Forcada, A. Meikle and J. A. Abecia, unpubl.
data). Melatonin improved embryo quality in control and
undernourished ewes (Va´zquez et al. 2009), which suggests that
melatonin may be part of a compensatory mechanism that
increases the quality of embryos in undernourished ewes by
ameliorating the oviduct milieu. To the best of our knowledge,
our study (Va´zquez et al. 2009) was the first to evaluate the
effects of the interaction between melatonin and nutrition levels
on the sheep endometrium and oviducts during the reproductive
and anoestrous seasons. Chuffa et al. (2011) reported that long-
term melatonin treatment in rats promoted differential regula-
tion of sex steroid receptors on reproductive tissues during
Melatonin and embryo viability in sheep Reproduction, Fertility and Development 85
ovulation, primarily acting in situ through the MT
1
receptor or
by altering the dynamics and responsiveness of sex steroid
receptor isoforms after binding to oestradiol or P4. Neverthe-
less, the regulation of sex steroid receptors in the uterus and
oviducts has not been fully elucidated.
Effects of melatonin on in vitro oocyte and embryo
maturation and fertilisation
Several studies have provided evidence that melatonin has a
direct effect on oocyte quality and development. Fu et al.
(2014) showed that melatonin (10
7
M) downregulated p53 and
upregulated Bcl2 and LH receptor (LHR) gene expression in
ovine granulosa cells under thermal stress. The p53 gene
product regulates maternal reproduction at the embryo
implantation stage (Hu 2009) and Bcl2 family members have an
important role in regulating apoptosis in female gonads (Kim
and Tilly 2004). Incubating germinal vesicle or MI stage human
oocytes (immature oocytes from superovulation cycles, which
are generally referred to as ‘rescue IVM’) with a range of
melatonin concentrations resulted in significant differences in
mRNA expression levels that provided a molecular basis for the
relatively higher quality of melatonin-treated versus untreated
IVM blastocysts (Hao et al. 2017). Exogenous melatonin
treatment had a positive effect on the quality of blastocysts
derived from those young oocytes, and regulated the p53 sig-
nalling pathway and the genes associated with DNA methyla-
tion during ‘rescue IVM’. In goats, the addition of 10
7
M
melatonin to the IVM medium improved the competence of
juvenile oocytes, which led to increased blastocyst production
of high-quality embryos. These outcomes were explained, in
part, by reduced ROS and the antioxidant effect of melatonin
(Soto-Heras et al. 2018).
Our experience in measuring the effects of melatonin on the
maturation, growth and development of oocytes has been
reported in several papers (Casao et al. 2009,2010a,2013). In
those studies, the oocytes from sheep ovaries collected from an
abattoir were assigned to one of four groups: three melatonin-
treated groups (10
6
,10
8
or 10
10
M melatonin) and one
control group. Matured oocytes were fertilised with fresh ram
semen and the embryos were cultured for 8 days, with melatonin
concentrations maintained throughout the IVF process. Melato-
nin treatment during IVM and IVF had no significant effect on
the maturation or cleavage rates; however, 10
8
M melatonin
significantly increased the blastocyst rate. We concluded that
the addition of low concentrations of melatonin to in vitro
embryo production media did not affect maturation and early
embryo development after IVF, although 10
8
M melatonin
increased blastocyst rate after 8 days in embryo culture (Casao
et al. 2009). In another experiment (Casao et al. 2010a), oocytes
were assigned to one of four groups, two of which were treated
with 10
5
or 10
6
M melatonin, and the other two groups
serving as untreated controls. After IVF with fresh ram semen,
the embryos produced in each group were cultured with or
without melatonin. The addition of melatonin to the IVM and
IVF media increased the IVM and early cleavage rates of sheep
oocytes, which could have been mediated primarily by the MT
1
receptor (Tian et al. 2017). In the study of Tian et al. (2017),
melatonin binding to the MT
1
receptor enhanced the expression
of bone morphogenetic protein (BMP) 15 (an oocyte-derived
growth and differentiation factor that is a critical regulator of
folliculogenesis in oocytes) and genes associated with cumulus
cell expansion, decreased cAMP levels in oocytes and enhanced
cGMP levels in oocytes and cumulus cells, which in part
regulates sheep oocyte maturation. Furthermore, we have dem-
onstrated that ovine cumulus cells have the biochemical machin-
ery to synthesise melatonin (Casao et al. 2013), and the
expression of genes encoding melatonin-synthesising enzymes
has been demonstrated in the cumulus cells of other species,
such as cattle (El-Raey et al. 2011). There is evidence that the
synthesis of extrapineal melatonin occurs in a variety of organs
that are not directly affected by circadian rhythms, with the
function of this melatonin seemingly primarily antioxidant and
antiapoptotic (Bubenik 2002). The melatonin synthesised in
cumulus cells accumulates in the preovulatory follicle, where it
acts to reduce the oxidative damage caused by the cumulus–
oocyte complexes (Tamura et al. 2012). To identify the mecha-
nism by which melatonin can affect oocyte maturation, we
performed an IVF experiment in which MT
1
and MT
2
receptor
antagonist were added to the maturation medium. The levels of
DNA damage in cumulus cells did not differ significantly
between oocytes exposed to exogenous melatonin and those in
the control group (Casao et al. 2013). IVM with luzindole, an
antagonist of MT
1
and MT
2
receptors, significantly increased
the proportion of cumulus cells that showed DNA damage, but
there was no significant difference in the extent of DNA damage
between control oocytes and those matured in the presence of
10 nM 4-Phenyl-2-propionamidotetralin (4-P-PDOT), an MT
2
receptor antagonist, suggesting that the protective action of
melatonin against DNA damage during IVM is mediated by
MT
1
receptors. Several studies have shown that membrane-
bound melatonin receptors mediate the protective effects of
melatonin against DNA damage in other cell types (Espino et al.
2011). In our studies, the addition of melatonin receptor antago-
nists to the maturation medium did not affect the maturation,
fertilisation or cleavage rates, but reduced the blastocyst rate
(Casao et al. 2010a), which suggests that the presence of
melatonin during oocyte maturation can improve embryo devel-
opment via MT
1
or MT
2
receptors.
In our first published account of the effects of melatonin on
embryo development in vitro (Abecia et al. 2002), we used
thawed embryos (46 morulae and 45 blastocysts) that were
cultured with or without melatonin. If the embryos were blas-
tocysts when the culture started, melatonin increased the pro-
portion that had hatched after 24 h of culture and reduced the
proportion of embryos at the end of the incubation period that
had degenerated. After cryopreservation, embryos are sensitive
to oxidative stress, which results in lipid peroxidation, mem-
brane injury and structural destruction. Succu et al. (2014)
assessed the effects of various concentrations of melatonin in
the post-warming culture on the ability of the ovine embryo to
restore its functions after cryopreservation. The beneficial
effects of melatonin on embryo development in the post-
warming culture occurred at the lowest concentration
(10
9
M) only, which suggests that at high concentrations
melatonin may be toxic to some degree towards preimplantation
86 Reproduction, Fertility and Development J.-A. Abecia et al.
embryos, as evidenced by an increase in the blastocyst apoptotic
index, and that the specific melatonin concentration to which
embryos are exposed is critical to obtain the desired effect.
Several hypotheses have been proposed to explain the
mechanisms involved in the effect of melatonin on embryo
development. Takada et al. (2012) observed that the proportion
of bovine cumulus cells during IVM that had no DNA damage
was significantly higher in the group maturated with melatonin
than it was in the control group. After IVF and embryo culture,
the cleavage and blastocysts rates in the treatment and control
groups were similar. Takada et al. (2012) concluded that
exogenous melatonin during IVM protects cumulus cells against
DNA damage, but it does not affect embryo development
in vitro.Tian et al. (2014) reported that melatonin supplemen-
tation significantly promoted oocyte maturation, and the devel-
opment of embryos and the mean cell number per blastocyst
produced after IVF improved substantially. Tian et al. (2014)
identified the expression of melatonin MT
1
and MT
2
receptor
genes in cumulus cells, granulosa cells and oocytes. Exposure to
melatonin receptor agonists and antagonists demonstrated that
the beneficial effects of melatonin on bovine oocyte maturation
are mediated through melatonin membrane receptors, and that
melatonin supplementation during bovine oocyte maturation
significantly upregulates the expression of some oocyte matu-
ration-associated genes (growth differentiation factor 9 (GDF9),
meiosis regulator and mRNA stability factor 1(MARF1), and
DNA methyltransferase 1a (DNMT1a)) and cumulus cell expan-
sion-related genes (pentraxin 3 (PTX3), hyaluronan synthase 1
(HAS1/2)), and that LHR1/2 and epidermal growth factor
receptor are involved in signal transduction and epigenetic
reprogramming (Tian et al. 2014).
The second hypothesised explanation for the improvement of
embryo quality when melatonin is present in the culture medium
is the ability of melatonin to reduce oxidative stress. A recent
review by Loren et al. (2017) presents evidence, in both humans
and domestic animals, that melatonin improves gamete and
embryo quality, protecting against oxidative and nitrosative
stress by scavenging free radicals, preventing the mitochondrial
pathway of apoptosis and inducing antioxidant enzyme activity.
Manca et al. (2014) demonstrated that plasma Trolox equivalent
antioxidant capacity (TEAC) measured monthly for 1 year was
significantly lower in pinealectomised than sham-operated
ewes. In the mouse, melatonin significantly improved the
quality of microinjected pronuclear embryos in culture,
increased the efficiency of embryo implantation and increased
the birth rate of pups by reducing oxidative stress and apoptosis
in the cultured microinjected pronuclear embryos (Tian et al.
2017). Melatonin is a direct scavenger of free radicals, an
indirect antioxidant and an important immunomodulatory agent
(for a review, see Kurutas 2016). Melatonin probably acts as a
free radical scavenger by donating electrons to the other com-
pound and thereby removing a variety of ROS, including the
highly toxic hydroxyl radical. Furthermore, melatonin stimu-
lates several antioxidative enzymes, including superoxide dis-
mutase (SOD), glutathione peroxidase (GPx), glutathione
reductase and catalase, and inhibits pro-oxidant enzymes such
as nitric oxide synthase, xanthine oxidase and lipoxygenase.
Moreover, evidence suggests that melatonin stabilises cellular
membranes, thereby helping them resist oxidative damage
(Kurutas 2016). In vivo, oocytes and embryos produce endoge-
nous ROS that have an important role as second messengers in
cellular functions via activation of cell signalling cascades.
However, excessive ROS can lead to serious consequences,
including DNA fragmentation, enzyme inactivation and cell
death (Hao et al. 2017). Wang et al. (2014) demonstrated
melatonin MT
1
and MT
2
receptor gene expression in bovine
MELATONIN
RAM
DONOR
EWE
embryo
IVC
IVF
RECIPIENT
EWE
oocyte
IVM
SPERM
PRESERVATION
Fig. 1. Melatonin can be included in multiple ovulation and embryo transfer (MOET) procedures and
assisted reproductive techniques (ARTs), namely IVM, IVF and in vitro culture (IVC): (1), insert one
melatonin implant in donor and recipient ewes 8–10 weeks before embryo collection or embryo
transfer; (2) insert three melatonin implants in rams at least 60 days before service or semen collection;
(3) melatonin can be included in the semen preservation medium to enhance sperm quality; (4)
melatonin can be added to the IVM, IVF and IVC media (10
6
–10
8
M) to improve oocyte maturation
and embryo development.
Melatonin and embryo viability in sheep Reproduction, Fertility and Development 87
embryos, and that melatonin treatment during bovine embryo
development significantly upregulated the expression of anti-
oxidative (GPX4,SOD1,BCL2) and developmentally important
(solute carrier family 2 (facilitated glucose transporter), member
1(SLC2A1), DNMT1A and desmocollin 2 (DSC2) genes, but
downregulated the expression of proapoptotic (P53,BAX and
caspase 3 (CASP3)) genes. The beneficial effects of the presence
of antioxidants in IVM or IVF culture media are not limited to
melatonin; other natural antioxidants, such as verbascoside, can
promote embryo development by protecting the oocyte against
oxidative stress during IVM (Martino et al. 2016).
Finally, melatonin regulates the complex embryo–fetal
developmental processes, inducing circadian rhythmicity in
the offspring and having a direct developmental effect on the
nervous and endocrine systems. Melatonin also protects the
foetal organs during development against damage from oxida-
tive stress (for a review, see Voiculescu et al. 2014).
Proposals for MOET and ART protocols including melatonin
In view of the positive effects of melatonin on embryo donor
and recipient ewes in vivo, ram seminal plasma and semen
quality, as well as the improved in vitro performance of oocyte
and embryo cultures, the inclusion of melatonin in the MOET
procedures and the laboratory ARTs, namely IVM, IVF and
IVC, could be an easy and efficient tool to improve these
programs. As an example, the following protocol is proposed
(Fig. 1):
8–10 weeks before embryo collection or embryo transfer,
insert one melatonin implant (Melovine/Regulin; CEVA
Sante´ Animale) to donor and recipient ewes
at least 60 days before service, insert three melatonin implants
in rams used for natural mating or semen collection for AI
to enhance sperm quality, melatonin can also be included in
the semen preservation medium because in vitro melatonin
treatment has been shown to decrease capacitation and
phosphatidylserine translocation at 1 mM and to increase
short-term capacitation at 100 pM, resulting in an increased
oocyte fertilisation rate after IVF (Casao et al. 2010c)
to improve oocyte maturation, 10
6
–10
8
M melatonin can
be added to the IVM medium, and similar melatonin con-
centrations can be maintained in the IVF and IVC media until
the blastocyst stage is reached.
Granulosa cells: melatonin receptors
p53 gene Bcl-2 and LHR gene
Cumulus cells: synthesize melatonin
protect DNA damage
Follicle: antioxidant activity
Embryos:
regulation DNA methylation genes
quality
P4 receptor population
PGF2α secretion
Uterus:
P4 secretionCorpus Luteum:
oocyte competence
2,3,4,5
7,8,9
oxidative stress
DNA damage
IN VITRO:
10,11
1
6
Fig. 2. Two ways by which melatonin exerts actions to protect fromoxidativestress or toimprove oocyte and embryo
quality (1) by reducing the oxidative stress caused by reactive oxygen species (ROS), and thereby preventing the
deterioration of oocytes and embryos; and (2) by affecting the mechanisms involved in the fertilisation process, from
ovulation to maternal recognition. Via the first mechanism, melatonin: (1) regulates follicle development and oocyte
competence,especially during the ovulatory process;(2) provides a similar protective effectin IVM and IVF procedures;
(3) stimulates several antioxidant enzymes; (4) inhibits pro-oxidant enzymes; and (5) stabilises cellular membranes. Via
the second mechanism, melatonin: (6) affectsthe growth pattern of the follicles and the steroidogenic capacity of the CL;
(7) protects cumulus cells against DNA damage during IVM; (8) blocks the generation of prostaglandin (PG) F
2a
by the
uterus;(9) induces differentialregulation of theprogesterone receptorand oestrogen receptorERain the endometriumand
oviducts;(10) regulates the expressionof some genes involved inembryo development and implantation; and (11)affects
blastocyst quality by regulating signalling pathways and the genes associated with DNA methylation.
88 Reproduction, Fertility and Development J.-A. Abecia et al.
Concluding remarks
Based on this review, we hypothesise that the beneficial effects
of melatonin on embryo quality are derived in two ways, which,
in turn, depend on whether melatonin is provided exogenously
or endogenously: (1) via a reduction in the oxidative stress
caused by ROS, which reduces the deterioration of oocytes and
embryos; and (2) effects on mechanisms involved in the ferti-
lisation process from ovulation to maternal recognition (Fig. 2).
Via the first mechanism, melatonin: (1) regulates follicle
development and oocyte competence through its protective
action, especially during the ovulatory process, when an exces-
sive amount of ROS is produced within the follicle; (2) provides
a similar protective effect in IVM and IVF procedures, when
oocytes and embryos can be affected by ROS; (3) stimulates
several antioxidant enzymes, including SOD, GPx, glutathione
reductase and catalase; (4) inhibits pro-oxidant enzymes such as
nitric oxide synthase, xanthine oxidase and lipoxygenase; and
(5) stabilises cellular membranes, which probably helps them
resist oxidative damage.
Via the second mechanism, melatonin: (1) affects the growth
pattern of the follicles and the steroidogenic capacity of the CL;
(2) by activating its receptors, protects cumulus cells against
DNA damage during IVM; (3) blocks the generation of PGF
2a
by the uterus; (4) induces differential regulation of the proges-
terone receptor and ERain the endometrium and oviducts; (5)
regulates the expression of some genes involved in embryo
development and implantation; and (6) affects blastocyst quality
by regulating signalling pathways and the genes associated with
DNA methylation.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
The authors thank Bruce MacWhirter for English language editing of the
manuscript.
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92 Reproduction, Fertility and Development J.-A. Abecia et al.
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    Objective: In this study, the effects of melatonin on superovulation and the transfer of transgenic embryos were investigated in Small-Tailed Han sheep. Design: Different doses of melatonin (0, 40 or 80 mg/animal) were subcutaneously implanted into both multiparous (4-5 years old) donors and recipients before superovulation and estrus synchronization. The one-year-old young ewes without melatonin treatment served to evaluate the reproductive efficiency of the adult multiparous ewes. Ewes with superovulation were used as embryo donors. The estrus were induced in embryo recipients after embryo transpimplanted. Results: The results showed that the number of corpora lutea of the ewes received subcutaneous 40 or 80 mg melatonin implant (13.4±1.05/ewe, 15.1±1.62/ewe) were significantly higher than that of in control group (8.8±0.37/ewe) (p<0.05). Similarily the number of recovered embryos from the ewes received subcutaneous 40 or 80 mg melatonin implant (10.3±0.84/ewe, 10.9±1.21/ewe) was significantly higher than the control group (6.2±0.60/ewe) (p<0.05). The transimplantd embryos from 40 or 80 mg melatonin treated donors dramatically improved the pregnancy and birth rates compared to control ewes. In addition, both 40 mg and 80 mg melatonin implatation lead to more lambs born per embryo. Conclusions: These observations provide valuable information for the application of melatonin in increasing superovulation and transgenic embryo transplantation efficiency in sheep.
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    The aim of this brief review is to clarify the role of melatonin in the production and preservation of mammalian gametes and embryos. Melatonin is an indoleamine synthesized from tryptophan in the pineal gland and other organs that operates as a hypothalamic-pituitary-gonadal axis modulator and regulates the waxing and waning of seasonal reproductive competence in photoperiodic mammals. A major function of the melatonin rhythm is to transmit information about the length of the daily photoperiod to the circadian and circannual systems in order to provide time-of-day and time-of-year information, respectively, to the organism. Melatonin is also a powerful antioxidant and anti-apoptotic agent, which is due to its direct scavenging of toxic oxygen derivatives and its ability to reduce the formation of reactive species. Mammalian gametes and embryos are highly vulnerable to oxidative stress due to the presence of high lipid levels; during artificial breeding procedures, these structures are exposed to dramatic changes in the microenvironment, which have a direct bearing on their function and viability. Free radicals influence the balance between oxidation-reduction reactions, disturb the transbilayer-phospholipid asymmetry of the plasma membrane and enhance lipid peroxidation. Melatonin, due to its amphiphilic nature, is undoubtedly useful in tissues by protecting them from free radical-mediated oxidative damage and cellular death. The supplementation of melatonin to semen extender or culture medium significantly improves sperm viability, oocyte competence and blastocyst development in vitro.
  • Article
    Two trials were undertaken to investigate the effects of treating seasonally anoestrous ewes with melatonin implants on date of first oestrus and other aspects of reproductive performance.Trial 1 involved a total of 368 Mule ewes and 79 Scottish Blackface ewes on five farms, approximately half of which were treated with a single subcutaneous implant of melatonin (Regulin®), containing 18 mg melatonin, between 23 July and 6 August 1986 and the remainder acted as untreated controls. Treatment had no significant effect on the date of first oestrus or conception rate in Mule ewes, although it increased the number of Scottish Blackface ewes mating (92% v. 73%) and the number of mated ewes conceiving (69% v. 54%) in a 5-week mating period, resulting in significantly more treated ewes lambing (63% v. 37%; P < 0·01). Litter size was higher in 4/5 flocks, although this only reached statistical significance in one Mule flock and the Scottish Blackface flock.A total of 2116 ewes from 17 commercial flocks were used in trial 2, approximately half of which were Suffolk/Suffolk-cross ewes and the remainder Mule/Mule-cross ewes. Implantation with melatonin occurred between 22 June and 24 July 1987. Flocks with over 100 ewes were divided into three equal-sized groups and treated with either 18 mg melatonin (one implant of Regulin, 36 mg melatonin (two implants of Regulin given at the same time) or acted as untreated controls. Flocks with less than 100 ewes contained only the 18 mg melatonin and untreated control groups. Treatment with melatonin significantly advanced the date of first oestrus in most flocks of both breeds (P < 0·05) but the magnitude of this effect was variable. Significant (P < 0·05 at least) increases in ‘potential’ (from scanning) mean litter size (+0·13 to +0·18) and actual mean litter size (+0·11 to +0·14) resulting from treatment with melatonin were apparent in ewes of both breeds when the data were pooled across all flocks, but only in 4/17 of the individual flocks.These results indicate that treatment with melatonin implants may be a simple and effective way of advancing the breeding season and enhancing litter size of early lambing flocks under commercial farming conditions in the United Kingdom, but treatment must be given >60 days before the start of the natural breeding season for benefits in date of first oestrus to be manifest.