Theriogenology 150 (2020) 280e287

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Theriogenology
journal homepage: www.theriojournal.com

Ovulation mechanism in South American Camelids: The active role of

b-NGF as the chemical signal eliciting ovulation in llamas and alpacas
Mauricio Silva a, b, Luis Paiva c, Marcelo H. Ratto c, *
a
Department of Veterinary Sciences and Public Health, Chile
b
Nucleus of Research on Agrifood Production, Universidad Cato
lica de Temuco, Temuco, Chile
c
Faculty of Veterinary Sciences, Universidad Austral de Chile, Valdivia, Chile

a r t i c l e i n f o a b s t r a c t

Article history: The ovulation-inducing effect of seminal plasma was first suggested in Bactrian camels over 30 years ago,
Received 21 January 2020 initiating a long search to identify the ‘ovulation-inducing factor’ (OIF) present in camelids semen.
Accepted 29 January 2020 During the last decade, primarily in llamas and alpacas, this molecule has been intensively studied
Available online 30 January 2020
characterizing its biological and chemical properties and ultimately identifying it as b-Nerve Growth
Factor (b-NGF). The high concentration of OIF/b-NGF in seminal plasma of llamas and alpacas, and the
Keywords:
striking effects of seminal fluid on ovarian function strongly support the notion of an endocrine mode of
Llama
action. Also, have challenged the dogma of mating induced ovulation in camelid species, questioning the
Alpaca
Ovulation
classical definition of reflex ovulators, which at the light of new evidence should be revised and updated.
Seminal plasma On the other hand, the presence of OIF/b-NGF and its ovulatory effect in camelids confirm the notion that
b-NGF seminal plasma is not only a transport and survival medium for sperm but also, a signaling agent tar-
geting female tissues after insemination, generating relevant physiological and reproductive conse-
quences. The presence of this molecule, conserved among induced as well as spontaneous ovulating
species, clearly suggests that the potential impacts of this reproductive feature extend beyond the
camelid species and may have broad implications in mammalian fertility. The aim of the present review
is to provide a brief summary of all research efforts undertaken to isolate and identify the ovulation
inducing factor present in the seminal plasma of camelids. Also to give an update of the current un-
derstanding of the mechanism of action of seminal b-NGF, at central and ovarian level; finally suggesting
possible brain targets for this molecule.
© 2020 Elsevier Inc. All rights reserved.

1. Introduction induced ovulators its occurrence is contingent upon copulatory

Mammalian species have been classified as either spontaneous
or induced ovulators based on the type of stimulus responsible for
eliciting GnRH release from the mediobasal hypothalamus [1]. In
spontaneously ovulating species (e.g., human, sheep, cattle, horse,
pigs), release of GnRH from the hypothalamus is triggered when
systemic estradiol concentrations exceed a certain threshold [2e5].
On the other hand, in reflex ovulators (e.g. rabbit, Bactrian camel,
llama, alpaca, cat, ferret) neural signals from copulatory stimulation
induce hypothalamic GnRH secretion, followed by the preovulatory
release of LH from the pituitary gland [1]. Similar to spontaneous
ovulators, a surge in LH appears to be requisite for ovulation, but in
* Corresponding author.
E-mail address: marceloratto@uach.cl (M.H. Ratto).
stimuli; hence, ovulation is not a regular cyclic event.
There appears to be an evolutionary pattern or preference for
induced versus spontaneous ovulation among species. Some have
suggested that induced ovulation is a primitive trait because of its
existence in primitive Orders like Rodentia, Lagomorpha and
Insectivora [6e8] but the presence of induced ovulators in the
Order Artiodactyla (e.g., llamas, alpacas and camels) is not consis-
tent with this theory. While there appears to be no phylogenic
pattern to the ovulatory mechanism [9], it is believed that envi-
ronmental factors or social cues influence the presence of induced
ovulation. Most recent additions to the list of induced ovulators
belong to the Order Rodentia [9].
Classification of species as spontaneous or induced may be
overly strict since induced ovulators can occasionally ovulate
spontaneously and ovulation in those considered spontaneous
ovulators can occasionally be induced or accelerated by copula.

https://doi.org/10.1016/j.theriogenology.2020.01.078
0093-691X/© 2020 Elsevier Inc. All rights reserved.
 M. Silva et al. / Theriogenology 150 (2020) 280e287
Indeed, recent pieces of evidence clearly indicate that the control of
the ovulatory process could be more complex that a simple copu-
latory stimuli to induce ovulation, in the case of reflex ovulators. It
has been well documented that an ovulation-inducing factor (OIF)
present in the seminal plasma of llamas, alpacas and Bactrian
camels is responsible to induce ovulation in these species. The
factor has been isolated and purified from the llama and alpaca
seminal plasma and it has been identified as Nerve Growth Factor
(b-NGF) [10e13].
As many reviews have recently been published [10e13]
describing the role of OIF or b-NGF in the mechanism of ovulation
in camelids, we believe that is relevant to present a retrospective
summary of all OIF/b-NGF studies that describe its effects on
ovarian function (i.e. ovulation and corpus luteum formation). In
addition, a brief update about the current knowledge of the pres-
ence, chemical identity and properties of OIF is given. Finally, the
potential brain targets for llama b-NGF as main sites to trigger
GnRH secretion are also suggested.
2. Unveiling the chemical induction of ovulation in camelids:
from OIF to b-NGF
The identification of b-NGF in the semen of most camelids
species and the clarification of its striking signaling action on the
female genital tract, ending in ovulation-induction, was achieved
after a long systematic search. Table 1 is intended to give a chro-
nological summary of the most relevant research undertaken in Old
and New World Camelids that contributed to this fascinating
discovery.
Several Chinese studies documented the first evidence for the
presence of an ovulation-inducing factor (OIF) in the Bactrian camel
semen 40 years ago (Table 1). Based on the results of these studies,
it was concluded that: a) a protein molecule present in the seminal
plasma was responsible for eliciting ovulation rather than the
mechanical stimulation of copulation [14,15]; b) ovulation induced
by the systemic or intrauterine administration of the factor is
preceded by a preovulatory LH surge [16]; and c) the factor is a
novel protein chemically different to GnRH, LH, hCG, eCG and
PGF2a [17]. However, all these studies failed to isolate, purify and
identify this factor from the seminal plasma of Bactrian Camel.
A study conducted years later in South American Camelids
(SACs) [18], alpacas and llamas, also documented the presence of an
OIF in the semen of these species because systemic administration
of homologous seminal plasma induced ovulation in 28 out of 30
treated females. Indeed, similar to the Bactria Camel studies,
ovulation was preceded by a preovulatory surge of LH [18]. Since
then, llamas and alpacas have emerged as a powerful animal model
to test the presence of OIF using either homologous or heterologous
seminal plasma [18e21], seminal fractions [22,23] or purified OIF
[24e26] after intramuscular, intravenous or intrauterine adminis-
tration. We want to point out that most of the information from the
initial studies presented in Table 1 refer to this protein molecule as
ovulation-inducing factor, OIF. This factor has now been identified
as the neurotrophin b-NGF in llamas, alpacas and Bactrian Camel
[27e30], Additionally, during the last 10 years this factor has been
biochemical and functionally characterized [18e20,22e25]. Based
on these studies, OIF has been described as a potent ovulatory
molecule in alpacas and llamas which also has a relevant luteo-
trophic effect in llamas [18,25,26,31,32], although, a similar luteo-
trophic effect has not observed in alpacas [28,33].
The primary source of OIF/b-NGF in llamas is the prostate gland
[34], the only accessory sex gland beside the bulbourethral gland in
male llamas. It is well known that b-NGF concentration in the
seminal plasma of llamas is much higher than that of spontaneous-
ovulating species (stallion and boar [35]; bull [36]). Also, it was
281
found that concentration of b-NGF is high in the seminal plasma of
another induced ovulator, such as the rabbit [37e39]. Further
studies, demonstrated that b-NGF represents approximately 50% of
total protein in the llama seminal plasma (12.4 mg/mL [23]), being
10 times more concentrated than in bovine semen [34]. Also b-NGF
has been described as one of the most abundant seminal proteins in
alpacas [28,29], accounting for 15e30% of total protein content of
seminal fluid [28,40].
3. Nerve Growth Factor (b-NGF): a neurotrophin beyond the
neuronal survival and protection
Neurotrophins (NTs) are proteins that promote differentiation,
growth and survival of many populations of peripheral (PNS) and
central nervous system (CNS) neurons during development and
adulthood [41e44]. The NGF is the best known and most studied
member of NT family including brain-derived NTs 3 4/5, 6 and 7
[45e47]. NGF is a neurotrophin conserved in species of sponta-
neous and induced ovulation and although its function was first
recognized at the level of PNS and CNS, several subsequent studies
have documented its effects in non-neuronal tissues including also
the ovary [48e50]. High and low affinity NGF receptors, TrkA and
p75 respectively, are found in the human ovary as early as the fetal
stage [48]. In rodents, NGF allows early follicular development and
ovulation [49,50] and ovary differentiation during development
[51].
Interestingly, roles for NGF have also been described in the
endocrine [52], immune [53,54], female [55,56] and male repro-
ductive systems [57]. NGF and TrkA are expressed by granulosa cells
in pre-antral and antral follicles [58] and appear to promote ovarian
development through initial differentiation of newly formed folli-
cles into gonadotropin responsive structures capable of ovulating
[59], as well as playing a pivotal role during ovulation [56]. In vitro
studies conducted in mice suggested that NGF contributes to the
ovulatory process inducing the secretion of estradiol, progesterone,
PGE2 and proliferation of theca cells [49]. It has also been docu-
mented that NGF and its receptors are expressed in granulosa cells
of antral follicles and in luteal cells of pigs and goats CL [60,61].
Furthermore, it has been suggested that the preovulatory LH surge
up regulates NGF and TrkA in the ovary [62,63], while NGF is
capable of regulating its own receptor in spontaneous ovulators
[64].
Lastly, the discovery of this neurotrophin at high concentration
in the semen of different spontaneous and induced ovulators,
mainly rabbit and camelids, contributed to confirm the notion that
seminal plasma is not only a transport and survival medium for
sperm but also, a signaling agent targeting female tissues after
insemination [65,66], generating physiological consequences as
relevant as ovulation induction.
4. Ovarian and endocrine effects of b-NGF
Systemic administration of purified NGF from llama seminal
plasma induces a higher and more sustained LH increase than that
observed after exogenous GnRH administration in female llamas
[18,24e26]. A significant increase in LH plasma concentration is
observed within 60 min after NGF treatment [18,25,67], similar to
that observed in females mated to an intact male [67]. Interestingly,
NGF LH-releasing effect was completely inhibited in llamas pre-
treated with GnRH antagonist Cetrorelix [25], which competitively
binds to GnRH receptors in the adenohypophysis. Therefore, it was
suggested that NGF elicits a preovulatory LH surge by a direct or
indirect action on GnRH hypothalamic neurons [25].
Additionally, using an ovariectomized llama model it was
demonstrated that the magnitude of LH secretion induced by NGF
282 M. Silva et al. / Theriogenology 150 (2020) 280e287

Table 1
Summary of main achievements in the discovery of ovulation-inducing factor (OIF/b-NGF) in the seminal plasma of camelids.

Old World Camelids: Bactrian camel (Camelus bactrianus)

Reference [Nº] Achievement

Chen et al., 1983 [14] - First report suggesting that seminal factors rather than mechanical stimulation of female genital tract are responsible for ovulation in
Bactrian camels.
Chen et al., 1985 [15] - Confirm that after vaginal artificial insemination (AI) seminal factors effectively induce ovulation in Bactrian camel.
- Suggest that ovulation-inducing factors (OIF) are present in seminal plasma and they are not related to sperm cells.
Xu et al., 1985 [16] - Show that as a consequence of vaginal AI with camel semen, circulating concentration of LH significantly rises after 4 h.
- Suggest that LH rise and consequently ovulation induction are evoked through a humoral pathway.
Zhao et al., 1990 [108] - Show that i.m. administration of camel seminal plasma induces ovulation in female camels.
- Suggest that OIF present in camel seminal plasma are proteins in nature.
- Suggest that OIF must reach the female pituitary gland via the blood stream to induce the LH surge.
Liu et al., 1995 [109] - Show that protein fractions isolated from camel seminal plasma can stimulate gonadotrophins secretion from in vitro cultures of rat, rabbit or
guinea pig pituitary cells.
Li et al., 2002 [110] - Demonstrate that i.m. administration of camel seminal plasma induce the release of LH in female camels.
Zhao et al., 2001 [106] - Describe the isolation of 5 protein fractions from Bactrian camel seminal plasma by DEAE-cellulose chromatography, one of which had LH-
releasing properties.
- Demonstrate that some of these seminal fractions can stimulate gonadotrophins secretion from in vitro cultures of rat pituitary cells.
- Demonstrate that i.m. administration of some of these seminal fractions induce the release of LH using female camels as an in vivo bioassay.
Pan et al., 2001 [17] - Demonstrate that OIF present in the seminal plasma of Bactrian camels is a novel protein different from GnRH, LH, hCG, eCG or PGF2a.
Li & Zhao 2004 [107] - Describe the isolation of 6 protein fractions from Bactrian camel seminal plasma by DEAE-cellulose chromatography, two of which had
in vitro and in vivo LH-releasing properties.
- Fail to purify the OIF present in the protein seminal fractions.
Druart et al., 2013 [29] - Identify Beta-Nerve Growth Factor (b-NGF) as the main protein in Bactrian camel seminal plasma.
Kumar et al., 2013 [30] - Describe the purification of b-NGF from Bactrian camel seminal plasma.

New World Camelids: Alpaca (Vicugna pacos)

Reference [Nº] Achievement

Paolicchi et al., 1999 [111] - First report suggesting the presence of a factor in alpaca seminal plasma that contributes to the secretion of LH and consequently to the
induction of ovulation in this species.
Adams et al., 2005 [18] - Report the existence of a potent OIF in the seminal plasma of alpacas and llamas capable of inducing a LH surge and ovulation.
- First evidence that OIF has also a luteotrophic effect in llamas.
Ratto et al., 2005 [19] - Suggest that OIF present in alpaca seminal plasma affects ovulation via a systemic rather than a local route.
- Demonstrate that curettage of endometrial mucosa facilitates the absorption of OIF.
- Demonstrate that ovulation in alpacas is not related to physical stimulation of the genital tract.
Ratto et al., 2006 [20] - Confirm the presence of an OIF in alpaca and llama seminal plasma.
- Suggest that OIF is a conserved molecule among mammals.
Kershaw-Young et al., - Identify the presence of b-NGF in high concentration in alpaca seminal plasma.
2012 [28] - Demonstrate that b-NGF is the seminal protein responsible for the induction of ovulation in alpacas.
Kershaw-Young et al., - Suggest that b-NGF is the seminal protein responsible for the induction of ovulation in alpacas.
2012 [40]
Druart et al., 2013 [29] - Confirm that b-NGF is the main protein in alpaca seminal plasma.
Stuart et al., 2014 [33] - Demonstrate that recombinant b-NGF at a dose of 1 or 0.1 mg is as effective as GnRH analogue (Busereline) for the induction of ovulation in
alpacas.
- Demonstrate that varying the dose of b-NGF does not affect timing of ovulation, size of corpus luteum (CL) or plasma progesterone
concentration in alpacas.

New World Camelids: Llama (Lama glama)

Reference Achievement

Adams et al., 2005 [18] - First report of the existence of a potent OIF in seminal plasma of alpacas and llamas capable of inducing a LH surge and ovulation.
- First evidence that OIF has also a luteotrophic effect in llamas.
Ratto et al., 2006 [20] - Confirm the presence of an OIF in alpaca and llama seminal plasma.
- Suggest that OIF is a conserved molecule among mammals.
Ratto et al., 2010 [22] - Demonstrate that seminal OIF is a protein resistant to heat and enzymatic digestion and has a molecular mass 30 kDa.
Ratto et al., 2011 [23] - Describe OIF as a 14 kDa seminal protein that elicits a LH surge, ovulation and the development of CL.
- Suggest an endocrine effect of OIF at hypothalamus or pituitary level.
Tanco et al., 2011 [24] - Demonstrate that OIF is found in high concentration in llama seminal plasma.
- Demonstrate that OIF presents a dose-dependent effect regarding induction of ovulation, CL diameter and plasma progesterone
concentration.
Bogle et al., 2011 [35] - Demonstrate that the mechanism for the biological response to OIF obtained from llama semen is also present in mice.
Bogle et al., 2012 [112] - Demonstrate that OIF induces LH secretion from in vitro cultured llama and bovine pituitary cells.
Ratto et al., 2012 [27] - Reveal that OIF present in llama seminal plasma is Beta-Nerve Growth Factor (b-NGF).
Silva et al., 2011 [37] - Show that rabbit seminal plasma induces a LH surge and ovulation in llamas but llama seminal plasma does not has the same effect in rabbit
does.
Silva et al., 2011 [25] - Demonstrate that GnRH antagonist (Cetrorelix) inhibits the preovulatory LH surge induced by OIF in llamas, strongly suggesting that LH
secretion is modulated by a direct or indirect effect of OIF on GnRH neurons in the hypothalamus.
Silva et al., 2012 [68] - Demonstrate that peripheral estradiol concentration partially modulates the effect of OIF on pituitary LH secretion; however, other(s) ovarian
factor(s) could also participate on this modulatory action in llamas.
Silva et al., 2014 [31] - Show that OIF induces larger CLs that secrete more progesterone when compared with GnRH in llamas.
Fern
andez et al., 2014 - Demonstrate that b-NGF exerts a potent luteotrophic effect which is dependent of administration frequency.
[102]
Ulloa-Leal et al., 2014 [26] - Demonstrate that b-NGF exerts its luteotrophic effect by altering the secretion of pituitary LH and enhancing vascularization of pre-ovulatory
follicle and early CL.
 M. Silva et al. / Theriogenology 150 (2020) 280e287 283

Table 1 (continued )

Old World Camelids: Bactrian camel (Camelus bactrianus)

Reference [Nº] Achievement

Silva et al., 2015 [114] - Show that regardless of route of administration (i.m., i.v. or intrauterine) b-NGF induces a LH surge, ovulation and the development of a
functional CL in female llamas.
- Demonstrate that the dose of b-NGF required by intrauterine route is higher than that of i.m. or i.v. routes.
Berland et al., 2016 [67] - Demonstrate that llamas can be induced to ovulate by seminal plasma b-NGF in the absence of copulation.
- Show that mechanical stimulation of copulation by itself is not able to induce ovulation in llamas.
- Demonstrate that promptly after copulation the concentration of b-NGF increases in general circulation in llamas.
Silva et al., 2017 [32] - Show that b-NGF increases CL vascularization and level of mRNA expression of steroidogenic enzymes during early stages of CL development
in llamas.
Silva et al., 2017 [113] - Suggest that the luteotrophic effect of b-NGF is influenced by administration route in llamas.
Bogle et al., 2018 [34] - Show that b-NGF is a common protein within the male accessory glands among camelids, bovids and cervids suggesting an important role in
the mechanism of ovulation in both induced and spontaneous ovulators.
Carrasco et al., 2018 [84] - Suggest that the ovulation-inducing effect of b-NGF in llamas is driven by an indirect interaction with GnRH neurons through hypothalamic
interneurons.
Valderrama et al., 2019 - Demonstrate that systemic administration of b-NGF purified from llama seminal plasma to female llamas induces a shift from estradiol to
[104] progesterone production in preovulatory follicles.
- Suggest that changes in gene expression patterns of steroidogenic enzymes observed in granulose cells of preovulatory follicles indicate an
ovarian local effect of b-NGF in this species.

is modulated by circulating concentration of estradiol [68], high-
lighting the role that the ovary has on the modulation of the hy-
pothalamus or pituitary response to NGF in this species. Moreover,
the administration of exogenous estradiol to ovariectomized llamas
was able to partially restore the magnitude of pituitary LH secretion
when compared to that observed in intact females, confirming that
plasma estradiol concentration affects pituitary LH secretion trig-
gered by NGF.
In a recent llama study [67], it was undoubtedly concluded that
the presence of b-NGF in semen is the pivotal factor responsible for
inducing ovulation, while penile stimulation during mating had no
effect. The results of this study showed that llamas mated with an
urethrostomized male did not ovulate (0/6), however ovulation rate
in llamas mated with an intact male or those given an intrauterine
infusion of seminal plasma was 83 (5/6) and 86% (6/7), respectively.
In addition, the results of this study indicated that intrauterine
administration of seminal plasma was sufficient to elicit the LH
surge, and that circulating LH concentration was positively corre-
lated with a rapid systemic increase in b-NGF observed during the
first hour after treatments (i.e. mating or intrauterine infusion of
seminal plasma). The results of these two complementary studies
[67,68] suggest that b-NGF from the seminal plasma is effectively
absorbed from the endometrium surface after copulation, rapidly
entering systemic circulation to elicit GnRH secretion from the
hypothalamus.
5. Potential brain targets of b-NGF eliciting ovulation
Once into the bloodstream, b-NGF could act at different pe-
ripheral targets, but central targets (brain tissues) are restricted
because of the blood-brain barrier (BBB). Nevertheless, several
evidences suggest that b-NGF may penetrate the brain or act in
brain areas lacking functional BBB [12,69] to stimulate the GnRH
preovulatory surge. In mammals, GnRH cells are crucial for repro-
duction, and yet their critical role is sustained by a remarkably low
number of cells [70,71] that are scattered in a bilateral continuum
in the basal forebrain [72]. Two major hypothalamic areas are
known to harvest a large number of GnRH cells: (i) the mediobasal
hypothalamus (MBH), and (ii) the preoptic area (POA) - anterior
hypothalamus [73]. Most of these cells display 2 long dendrites [74]
from which many axon-like projections emerge (so-called ‘den-
drons’) [75] directing to the median eminence (ME) where their
nerve endings are close to fenestrated vessels of the hypophyseal
portal system that transport GnRH molecules to the anterior pitu-
itary [76,77]. Several pieces of evidence indicate that the preovu-
latory LH secretion is commanded by a subpopulation of GnRH cells
located in the anterior hypothalamus in different species. Approx-
imately 40% of the GnRH cells located in the POA and anterior hy-
pothalamus express Fos protein, a widely used marker for neuron
activation, in the late proestrus preceding the LH surge in rats [78]
and mice [79,80]. Similarly, in ferrets, also an induced ovulator,
mating induces the expression of Fos in 50% of the POA and anterior
hypothalamus GnRH cells, but also in the MBH [81,82].
Like in other species, the llama MBH and the anterior hypo-
thalamic area harvest the two largest populations of GnRH cells,
exhibiting the former twice the number of this type of neurons
than that observed in the latter [83]. In this species, GnRH cells
were also detected in the POA and the diagonal band of Broca [83].
Interestingly, in llamas, the diagonal band of Broca displays the
highest number of undifferentiated cells (i.e. not GnRH neurons)
expressing the high-affinity b-NGF receptor TrkA, although this
expression is also high in the POA and anterior hypothalamus,
whereas the MBH exhibits the lowest number of cells expressing
this receptor. The low-affinity b-NGF receptor p75 showed a similar
distribution pattern [84]. In the same llama study [84], expression
of TrkA, but not p75, is reported to occur in a low number (less than
3%) of GnRH cells. Interestingly, according to Herbison [85], the
recruitment of a small number of GnRH cells usually involved in
GnRH or LH surges (10% in sheep and 20% in mice, respectively) is
sufficient to elicit ovulation. Hence, it could be that, as in other
species, the llama LH preovulatory release may be elicited by acti-
vation of GnRH cells located in the rostral hypothalamus. Whether
b-NGF is able to penetrate the brain in physiologically significant
amounts has not been determined yet.
Interestingly, a recent mice study [86] supports the notion that,
in llamas, b-NGF could modulate the activity of GnRH cells by
activation of central receptors. In a male mice model of hypo-
gonadotropic hypogonadism, intracerebroventricular administra-
tion of b-NGF: a) increased plasma LH (and FSH) in vivo; b)
stimulated GnRH secretion from hypothalamic GT1-7 immortalized
cells (expressing TrkA receptors) in vitro and c) reversed the
hypogonadism as increased gonadal weight and plasma testos-
terone. This evidence suggests that cellular and molecular mecha-
nism of b-NGF signaling on GnRH systems are present in other
species and are not sex-specific.
284 M. Silva et al. / Theriogenology 150 (2020) 280e287
5.1. Kisspeptin neurons
Even though the role of GnRH neuroendocrine cells orches-
trating fertility remains undoubted, it is established that, in most
animal species, these cells lack the estrogen receptor alpha (ERa)
required to trigger the preovulatory GnRH surge by gonadal ste-
roids in spontaneous ovulators [87]. In the last decades, kisspeptin
neurons which express ERa have emerged as the main input con-
trolling the activity of GnRH cells by two hypothalamic populations
located in the arcuate nucleus and the POA. So when estradiol
concentration increases, Kiss1 gene expression in cells of the
arcuate nucleus is downregulated, whereas it is upregulated in the
POA eliciting the preovulatory GnRH surge [88]. In the induced
ovulator musk shrew (Suncus murinus), the expression of Kiss1
seems to be controlled in a similar fashion as in spontaneous
ovulators, but the copulatory stimulus is reported to be the final cue
that activates POA kiss1 cells by means of c-fos expression [89];
however, if this leads to activation of GnRH cells warrants further
investigation. Recently, kisspeptin has been proposed as the po-
tential mediator of the b-NGF-stimulated ovulation in camelids
[13]. Interestingly, in a preliminary llama experiment, the ovulatory
effect of the i.v. administration of kisspeptin-10 (the short biolog-
ically active murine analogue) is interpreted as evidence for the
involvement of kisspeptin in camelids ovulatory pathway under the
assumption that kisspeptin-10 acts on the brain [90]. Small pep-
tides, such as kisspeptin-10, are often believed to penetrate the
BBB, although other hypothalamic peptides of similar molecular
weights (~1000 g/mol), like oxytocin, do not, at least not in
neuroactive amounts [91]. In mice, i.v. kisspeptin-10 has failed to
activate GnRH cells (as measured by Fos expression), and yet both
short and long kisspeptin forms stimulate LH release and depen-
dent effect [92,93], indicating that systemic kisspeptin acts outside
the BBB (e.g. pituitary [94,95]; ME [96]). The detection of kisspeptin
neurons in the llama brain and whether they express receptors for
b-NGF (i.e. TrkA, p75) awaits to be shown to propose potential
physiological roles.
5.2. Circumventricular organs (CVOs)
On the other hand, circumventricular organs (CVOs) are small
neurohemal brain areas lacking functional BBB due to the presence
of fenestrated capillaries allowing neurons to monitor and sense
compounds present in systemic circulation (i.e. sensory CVOs), and
also the release of neuroendocrine products into the bloodstream
(i.e. secretory CVOs) [97]. Two CVOs are closely linked to the GnRH
system and so are likely targets for circulating b-NGF in llamas: (i)
the organum vasculosum of the lamina terminalis (OVLT) which
contain sensory neuronal elements, and (ii) the ME where GnRH
nerve endings release their product into the portal circulation.
Consistent with the above, Loy [69] reported intense labeling of
both CVOs following the i.v. administration of 125I-b-NFG in rats.
In mice, GnRH cells of the rostral POA extend their dendritic
processes into the OVLT, and even some GnRH somas (located
within 100 mm from OVLT) are reported to be exposed to com-
pounds present in the bloodstream; 85% of these neurons
expressed Fos in response to E2-induced LH surge, and locally
applied kisspeptin to some of these cells induced excitatory re-
sponses [98]. So, it might be that GnRH cells expressing TrkA re-
ceptors [83,84] are in contact with b-NGF reaching the OVLT
following copulation.
In the ME, b-NGF labeling has been reported to occur in cells of
this CVO in seminal plasma-treated llamas [12], and rats treated
with an antibody against NGF showed blunted GnRH release from
the ME in response to application of PGE2 in vitro [99], suggesting a
potential role for b-NGF modulating the GnRH release from this
structure.
Finally, noradrenergic inputs arising from A2 cells of the caudal
brainstem are known to modulate the activity of preoptic GnRH
cells at the time of the LH surge [100], and noradrenaline is thought
to play a major role in the rabbit reflex ovulation [101], however, a
recent study [67] has ruled out the contribution of neural pathways
arising from the reproductive tract on the llama ovulatory
mechanism.
6. Luteotrophic effect of b-NGF
The preovulatory LH surge is the key signal for inducing not only
ovulation but also the initial stages of luteinization, CL formation
and functioning. We have previously documented that seminal
plasma b-NGF is a protein with a significant luteotrophic effect in
llamas [18,23e26]. From early stages of CL development, induced
by intramuscular administration or by intrauterine infusion of b-
NGF, this gland consistently secreted more progesterone than those
induced after GnRH treatment. In all these previous studies, there
was a positive relationship between the magnitude of LH release,
and subsequent luteal function when females were treated with
seminal plasma or purified seminal plasma b-NGF vs GnRH. These
pieces of evidence have provided a rationale for the hypothesis that
LH may mediate the luteotrophic effect of seminal plasma b-NGF. In
a recent llama study [26] the use of power doppler ultrasonography
demonstrated that the preovulatory follicle and the early CL has a
greater vascularization in seminal plasma b-NGF- than in GnRH-
treated females, resulting in higher progesterone secretion.
Indeed, further studies confirmed that luteotrophic effect of semi-
nal plasma b-NGF was associated with enhanced tissue vasculari-
zation during the peri-ovulatory period and early stages of CL
development [26,102]. This could be related to an increase of the
vascular endothelial growth factor (VEGF) which induces angio-
genesis by stimulating the proliferation of endothelial cells of
preexisting capillaries [103]. In addition, the increasing of CL
vascularization was confirmed in a previous llama study [32],
where females treated with seminal plasma b-NGF displayed a
greater CL vascularization area and up-regulation of mRNA
expression of p450SCC and StAR in luteal tissue, at Day 4 and 8 of
luteal phase enhancing progesterone secretion. Recently, we [104]
found that mating and systemic administration of purified llama b-
NGF induced a rapid shift from estradiol to progesterone produc-
tion in the preovulatory follicle in llamas. In these females there
was an up-regulation of the main genes related to progesterone
production resulting in an increase of P4/E2 ratio in the follicular
fluid. Llamas mated or treated with b-NGF displayed significant
higher changes in gene expression patterns of steroidogenic en-
zymes than that observed in GnRH-treated females, suggesting a
local effect of systemic b-NGF at ovarian level. In this sense, we
demonstrated that systemic concentration of NGF and LH increased
during the first 3.5 h after administration of seminal plasma b-NGF
[67] which could be related to a local effect of seminal plasma b-
NGF at ovarian level stimulating progesterone production. In fact,
the addition of purified llama b-NGF in the in vitro culture of llama
granulosa cells increased the mRNA relative expression of StAR and
VEGF resulting in higher progesterone secretion than that observed
in granulosa cells treated with LH after 48 h of in vitro culture [104].
7. Final remarks
As demonstrated, the high correlation between the concentra-
tion of b-NGF, in llama and alpaca semen, and its direct effects on
ovarian function support the notion of an endocrine mechanism
controlling ovulation in these species, which is not as a response to
physical stimulation of the female genital tract itself. Thus, in these
 M. Silva et al. / Theriogenology 150 (2020) 280e287
2 species ovulation is a direct consequence of b-NGF absorption
from the genital mucosa after mating, its rapid increase and sys-
temic distribution to distant target tissues, the increase in hypo-
thalamic GnRH release, the surge in circulating concentrations of
LH, and finally a series of molecular, endocrine and morphological
changes at follicular level ending in the release of the oocyte and
the development of a CL.The above clearly set the foundations for
the establishment of a new category of induced-ovulators.
Although substantial advances have been made during the last
decades to reveal the exact mechanism controlling ovulation in
llamas and alpacas, there is still missing information and several
research gaps need to be addressed before full comprehension of
this phenomena is acquired. According to the authors, the most
relevant research gaps still existing are:
a) The identification of brain targets from which b-NGF can
start a neuronal chain reaction ending with the hypotha-
lamic GnRH release. In this regard, the main questions still
remaining unanswered are:i) how does b-NGF reaches GnRH
neurons located inside de Blood-Brain Barrier (BBB)?, ii) is
there any possibility for b-NGF to affect target tissues outside
the BBB to trigger its effect?, and iii) what are the compo-
nents of the intermediary neuronal network translating
initial action of b-NGF on tissue target and its final effect on
GnRH neurons?
b) The determination of a direct ovarian local effect of b-NGF on
follicular cell populations able to induce ovulation, or at least
to complement its central action at hypothalamic-
hypophyseal axis. The study of such a local effect using and
in vivo model represents a great challenge since discrimi-
nation of b-NGF actions at both levels (central vs ovarian) is
extremely difficult in a confounding physiological environ-
ment proper of this model.
c) The abundance of b-NGF in seminal plasma of llamas, alpacas
and camels is an extraordinary reproductive feature that
probably has other implications besides ovulation induction
in these species. Thus, how the high concentration of this
seminal protein relates to male reproductive characteristics,
such as sperm physiology or fertility; or how may affect other
female reproductive traits, once it reaches the genital tract
after copulation, are relevant subjects yet unexplored.
d) Although there is a direct relationship between both wild
and domestic species of South American Camelids through
the domestication process (i.e. llamas and alpacas evolved
from guanacos and vicugnas respectively), there is no infor-
mation regarding the ovulation mechanism prevailing in
wild SACs, neither if the process of domestication altered
ovulation pattern present, as it has affected several other
reproductive characteristics in many animal species [105].
Acknowledgements
Several studies described in the present review were supported
by the Chilean Council of Science and Technology (CONICYT, Fon-
decyt: 1160934, 11140396).
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