The timing of puberty ( oocyte quality and management )

This review aims at giving an overview on the physiological events leading to puberty onset in mammals and more specifically in cattle. Puberty is an important developmental milestone in mammals involving numerous changes in various physiological regulations and behaviors. It is a physiological unique event integrating several important central regulations at the crossroad of adaptation to environment: reproductive axis, feeding behavior and nutritional controls, growth, seasonal rhythm and stress. Puberty onset is also an important economic parameter in replacement heifer program and in genomic selection (genomic bulls). The quest for advanced puberty onset should be carefully balanced by its impact on physiological parameters of the animal and its offspring. Thus one has to carefully consider each step leading to puberty onset and set up a strategy that will lead to early puberty without being detrimental in the long term. In this review, major contributions in the understanding of puberty process obtained in rodents, primates and farm animals such as sheep and cattle are discussed. In the first part we will detail the endocrine events leading to puberty onset with a special focus on the regulation of GnRH secretion. In the second part we will describe the neural mechanisms involved in silencing and reactivating the GnRH neuronal network. These central mechanisms are at the crossroad of the integration of environmental factors such as the nutritional status, the stress and the photoperiod that will be discussed in the third part. In the fourth part, we will discuss the genetic determinants of puberty onset and more particularly in humans, where several pathologies are associated with puberty delay or advance and in cattle where several groups have now identified genomic regions or gene networks associated with puberty traits. Last but not least, in the last part we will focus on the embryologist point of view, how to get good oocytes for in vitro fertilization and embryo development from younger animals.


Introduction
Puberty is an important developmental milestone in mammals involving numerous changes in various physiological regulations and behaviors.It is a physiological unique event integrating several important central regulations at the crossroad of adaptation to environment: reproductive axis, feeding behavior and nutritional controls, growth, seasonal rhythm and stress.

Puberty, puberty onset, peri-pubertal, reproductive maturity what's the difference
Puberty onset results from a complex and integrated sequence of biological events leading to progressive maturation of sexual characteristics that ultimately lead to attainment of full reproductive capacity.This sequence is referred as the timing of puberty.Puberty timing in mammals is the result of evolution allowing females to attain ideal pelvic anatomy and size, complete growth and maximize skeletal mineralization, prior to the demands of pregnancy, lactation and offspring rearing.
Puberty is defined as the moment of the first emission of gametes, ie the first ovulation in females and the first spermatozoa entering the epididymis in males.Therefore puberty is expressed as a date or as an age.From this definition, it is obvious that puberty can be easily detected in females by detecting the first ovulation.However in males there is no non-invasive method to assess the presence of epididymal spermatozoa and it is usually defined according various physical and behavioral changes.Therefore puberty is very often studied through the modifications observed before and immediately following the first emission of gametes.In that case it is better to speak of peri-pubertal period.For example in females, breeders usually monitor the exterior signs of receptivity (age at first estrus).However one has to keep in mind that estrus behavior can exist without a proper ovulation and the reciprocal is also true: ovulation can occur without any sign of estrus behavior.For males, breeders look at the sexual behavior too: mounting behavior and erection.Here again, this behavior does not mean that is there any spermatozoa in the ejaculate.
The strict definition of puberty onset as the first emission of gametes does not mean that the animals are able to breed yet.They can produce and release gametes but reproduction is more than that.Females usually need a period of time after puberty onset to have regular ovarian cycles and to get their uterus capable of supporting a pregnancy.For males, the concentration of spermatozoa in the ejaculate should reach a certain threshold to give an adequate fertility; here again this can take some time after the puberty onset.Reproductive maturity is another phenomenon and the mechanisms leading to puberty onset are different from those leading to reproductive maturity.Anim.Reprod., v.13, n.3, p.313-333, Jul./Sept.2016

Brief overview of the endocrine events across the estrus cycle
Post-pubertal females present estrus cycles, which is the reflection of the ovarian cyclicity.During the late follicular phase, the preovulatory follicles release high estradiol levels in the blood stream.The starting point of all endocrine events leading to ovarian cyclicity is the secretion of a neurohormone: the gonadotropin releasing hormone (GnRH).GnRH acts on the gonadotrope cells located in the anterior pituitary and promote the synthesis and release of both gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH).LH and FSH will act at the ovary level to promote the release of gonadal steroid estrogens and progestagens and to promote follicular growth.Granulosa cells and thecal cells collaborate to synthesize and release estrogens, among which 17-βestradiol (E2) is the most prevalent estrogen in most species.The amount of estrogen released is dependent on the number of granulosa and thecal cells.Considering the growth of a sphere, the amount of estrogen that can be synthetized is proportional to the cubic of the follicle radius.Therefore the final growth of the dominant follicle is accompanied by a huge increase in E2 production.High E2 levels are responsible for the expression of estrus behavior.In most species studied, high E2 levels will also exert a positive feedback on GnRH secretion leading a large amount of GnRH release that causes a large amount of LH release: the pre-ovulatory GnRH and LH surges (see Fig. 1).LH surge occurs just before the ovulation and last several hours after, contributing to the luteinization of granulosa and thecal cells of the ovulated follicle.De facto, E2 levels drop and the positive feedback disappears, stopping its repressive action on GnRH secretion.In parallel progesterone (P4) secretion increases and exerts a negative feedback on GnRH secretion.High P4 levels have a positive action on E2 receptors (ERs) expression.Without P4-priming during the previous cycle, ERs expression is low and despite high E2 levels during the preovulatory phase, the estrus behavior, which is strongly dependent on ERα signaling, is poorly expressed.Once luteolysis occurs, P4 levels drop, the negative feedback is suppressed and GnRH secretion increases again leading to LH and FSH release and a new follicular phase starting.

Evolution of gonadotropin secretion in the pre-pubertal period
The key decisive event required for puberty to occur is an increase in pulsatile gonadotropin releasing hormone (GnRH) release from GnRH neurons leading to gonadotropins LH and FSH secretion.In mature adult, the GnRH is released in the portal veins in a pulsatile manner.GnRH secretion is difficult to assess.As a matter of fact, GnRH is released in capillaries within the ME that form portal veins along the pituitary stalk.From these portal veins, pituitary capillaries emerge and the GnRH is released in the intercellular space and reaches anterior pituitary cells.GnRH concentration in the portal veins varies between 4-100pg/ml (Caraty et al., 1982;Clarke and Cummins, 1982;Levine et al., 1982;Irvine and Alexander, 1987;Gazal et al., 1998), which gives a very small amount of GnRH for the small blood volume considered.Thus, the amount of GnRH that passes in the general circulation is very small; the concentration is well below the detection threshold of known hormonal assays.Moreover GnRH half-life is very short, a few minutes.Due to its small peptidic structure, circulating endopeptidases degrades rapidly the GnRH.Therefore to assess the GnRH secretion, blood should be punctured from pituitary portal vessels or from canulae inserted in the third ventricle (Gazal et al., 1998) and this can only be performed in large animals and requires invasive surgical procedures (Clarke and Cummins, 1982;Levine et al., 1982).An alternative is to follow LH secretion since it has been clearly demonstrated that a GnRH pulse precedes every LH pulse (Clarke and Cummins, 1982;Caraty et al., 1989).
In the female Rhesus monkey the early prepubertal period is characterized by an increase in pulsatile release with a concomitant increase in pulse frequency and pulse amplitude.In the midpubertal phase, only an increase in GnRH pulse amplitude is noticed and the global GnRH secretion is increased during the night (Watanabe and Terasawa, 1989).This is in contrast to ewes and heifers where the midpubertal period is characterized by an increase in LH pulse frequency associated with a decrease in pulse amplitude (Day et al., 1987).In heifers, the frequency of LH pulses is usually in a range of 2 to 4 pulses/24 h 100 to 50 days before puberty onset.The amplitude of LH pulses is high, reaching 6-8 ng/ml.From 50 days before to puberty onset, the frequency of LH pulses increased to reach 15-20 pulses/24 h and the mean amplitude of LH pulses decreased to values (<2 ng/ml; Day et al., 1987).Such increase in LH pulse frequency was also reported in female lambs (Claypool and Foster, 1990).In humans, this increase in pulsatile LH secretion is also observed but occurs during the night phase (Wu et al., 1996).
In spite of numerous physiological studies in model animals, little is known about the key events leading to GnRH neurons progressive activation at puberty onset.The scientific community admits that puberty onset is preceded by gradual changes in transsynaptic and glial inputs to the GnRH neuronal network.The trans-synaptic changes consist of a coordinated increase in excitatory inputs and/or a reduction in inhibitory influences.Glial cells could also participate in regulating extracellular glutamate concentration, and in releasing growth factors and small diffusible molecules that directly or indirectly stimulate GnRH secretion.In addition to the classical excitatory glutamatergic neurons, kisspeptin signaling through GPR54 was discovered in 2005 as a powerful stimulator of GnRH release (Messager et al., 2005).Nevertheless, how these key events are triggered through environmental and nutritional factors is far from being understood.
Figure 1.Schematic representation of the hypoyhalamic-hypophysis-ovary axis.GnRH neurons somas are mostly located in the preoptic area and send their axons towards the median eminence where GnRH is released in a pulsatile manner into capillaries.Median eminence capillaries merge to form the portal vessels on the ventral part of the anterior pituitary and give rise to pituitary capillaries.GnRH then can diffuse within the anterior pituitary and reach gonadotropic cells that release the gonadotropins: LH and FSH.LH and FSH will reach the general blood circulation and act on the ovaries to stimulate both oocyte and follicle growth and gonadal steroids secretion.Progesterone exerts an negative feedback at the pituitary and hypothalamic levels, estradiol at low concentration exerts a negative feedback at both pituitary and hypothalamic levels, but at high concentration (during estrus) it will have a positive feedback at both pituitary and hypothalamic levels.

GnRH control
The two modes of secretion GnRH secretion is characterized by two modes of secretion: pulsatile and continuous (the surge).These two modes have been described in the pioneering work of Ernst Knobil in the rhesus monkey where he described a tonic and a phasic mode of LH secretion controlled by two different areas within the hypothalamus Preoptic area (POA) and mediobasal hypothalamus (MBH), respectively (Nakai et al., 1978).The pulsatile pattern of GnRH secretion was confirmed in the 80's when a trans-nasal surgical approach allowed the collection of blood from the portal vessels between the hypothalamus and the pituitary (Clarke and Cummins, 1982;Levine et al., 1982).
GnRH/LH secretion is pulsatile during the follicular and the luteal phases, the surge mode occurs during the pre-ovulatory period.In most species where GnRH and LH secretions have been monitored simultaneously, the LH secretion profile is a good estimate of the GnRH pulsatile secretion: a GnRH pulse always precedes one LH pulse.The frequency of pulsatile secretion varies across the estrus cycle.For example in the ewe, the follicular phase is characterized  (Moenter et al., 1991).The GnRH pulse frequency is decoded by the GnRH receptor (GnRH-R) expressed by gonadotropic cells: high frequency favors the expression of the β-LH subunit whereas low frequency favors the expression of the β-FSH subunit (Bédécarrats and Kaiser, 2003;Thompson and Kaiser, 2014).

Anatomy of the GnRH neuronal network
The GnRH is a small peptide (10 amino-acids) issued from the processing of pre-pro-GnRH encoded by the Gnrh1 gene.The pre-pro-GnRH is processed in GnRH neurons to give the GnRH and the GnRHassociated peptide (GAP), Both are packed in large dense core vesicles (LDCV) for further release (Clarke et al., 1987).The GnRH is synthetized and secreted by a specialized population of neurons: the GnRH neurons.In most mammals the GnRH neurons' somas are located in the POA with a few cell bodies located in the MBH and the axons project towards the median eminence at the bottom of the MBH.However in primates, the repartition is different with the majority of GnRH neurons' somas located in the MBH and just a few in the POA.Axonal projections are projected to the median eminence where GnRH is released in blood capillaries and transported in portal vessels to the capillaries network of the anterior pituitary where it will stimulate the expression and release of the gonadotropins FSH and LH.

Extracerebral embryonic origin of GnRH neurons
During embryogenesis, the GnRH neurons originate from the medial part of the nasal embryonic placode at early embryonic age 30 (E30) in sheep (Caldani et al., 1987;Caldani et al., 1995), E11.5 in mouse (Schwanzel-Fukuda andPfaff, 1989;Wray et al., 1989), 6-7weeks of pregnancy in humans (Schwanzel-Fukuda et al., 1996).They then migrate along olfactory-, vomero-nasal-and terminal nerves to finally enter the forebrain through the cribiform plate.In the sheep, this phase of nasal migration is completed at E45, in the mouse at E13.5.Once in the brain they turn ventrocaudally to reach their final location in the POA/MBH.The phase of intra-cerebral migration is completed at E60 in the sheep and E16.5 in the mouse.Once settled, they grow their axonal projections toward the ME.This phase of axonal growth is terminated at E70 in sheep and E18.5 in the mouse.Once connected to the ME, it is believed that GnRH secretion occurs since it is correlated with the first observation of β-LH expression in the pituitary cells (Messaoud-Toumi et al., 1993).Primary cultures derived from embryonic nasal explants from E26 sheep embryos, E35 rhesus monkey embryos or E11.5 mouse embryos allow the development of functional secreting GnRH neurons that form a network in vitro.The GnRH secretion is pulsatile and the frequency is correlated to what it is observed in vivo according to each species considered (Duittoz and Batailler, 2000;Constantin et al., 2009).These in vitro approaches suggest that the pulsatility of secretion is an endogenous property of the GnRH network and that this property develops during the fetal life.

Functionality of the fetal GnRH neuronal network
Whether the pulsatile secretion develops in utero and plays a role in development has been studied particularly in the sheep species.Several groups have carried on a series of experiments on sheep fetuses.In chronically catheterized ovine fetuses, both LH and FSH exhibit a similar trend of peak values in midgestation (70-100 days) with a progressive decrease in plasma concentrations towards term (145 days; Sklar et al., 1981).Measurements from 55-60 days of gestation embryos gave low values of plasma LH and FSH concentrations.This pattern is similar to the one described in human fetuses with high concentration values of LH between 15-29 weeks of gestation (Kaplan and Grumbach, 1976;Clements et al., 2009).The ovine fetal pituitary gland has the capacity to respond to exogenous GnRH as early as 60 days of gestation with a maximal amplitude occurring during mid-gestation (Mueller et al., 1981).The pulsatile nature of LH fetal secretion was clearly assessed by serial blood sampling during a 4 h period in ovine fetuses at mid-gestation (Clark et al., 1984;Fig. 2).If we put in parallel the physiological maturation profiles of LH secretion and the development of the GnRH neuronal network we can clearly see the correlation between those events.Thus, once GnRH neuronal migration and axonal growth toward the median eminence is completed, the GnRH secretion can take place and induce the expression of gonadotropin subunits (Messaoud-Toumi et al., 1993).LH and FSH will act on the fetal gonad to stimulate gonadal steroid synthesis, and this is particular evident in male ovine fetuses where a spurt in testosterone secretion is detected at mid-gestation.This increase in testosterone in male fetuses or new born has been demonstrated in numerous mammals (Foster and Hileman, 2015;Plant et al., 2015;Prevot, 2015).In precocious mammals such as ovine and bovine species, the spurt occurs during the last third of gestation and is terminated at birth, whereas in altricial species such as rodents, the spurt occurs during the last days of pregnancy and during the first week post-natal (mice; Sisk and Foster, 2004).After this "mini puberty", the frequency of GnRH/LH secretion dramatically decreases and steroid levels drop; the infancy period is starting.
Figure 2. Schematic representation of the evolution of LH secretion from fetal life to adulthood in the ovine species.At 26 days of gestational age (G26), the first GnRH neurons are detected in the medial part of the nasal placode.From G26 to G35, GnRH neurons are born in the nasal placode and migrate along the nasal septum to reach the cribriform plate (intra nasal migration).From G35 to G45, GnRH neurons migrate into the brain and reach their final location in the preoptic area.From G45 to G60, GnRH neurons send axonal projections towards the external part of the median eminence (Caldani et al., 1995).At G60, the first expression of LHB is detected, suggesting that GnRH secretion is functional.From G80 to G120, LH is released in a pulsatile manner and contributes to the secretion of testosterone in male fetus and the sexualization of external genitalia and brain structures.From G120 to postnatal day 60 (PN60) there is virtually no LH secretion.From postnatal week 10 (PNW10) to PNW20, LH pulsatile secretion reappears with low frequency and high amplitude.From PNW20 to PNW30, the frequency of LH pulses increases and the amplitude decreases.The first preovulatory LH surge signs the onset of puberty.

Puberty: endocrine or brain revolution?
The pubertal transition involves both gonadal and behavioral maturation.The increase in the frequency of GnRH release and gonadotropins secretion progressively leads to the onset of gonadal functions: gametogenesis and steroid production.These steroids act in turn onto the brain to remodel neural circuits particularly those involved in sexual behaviors, but not only (Forger et al., 2015).In humans, several neurological or psychiatric diseases appear or are exacerbated at puberty (autism, schizophrenia, epilepsy, anorexia nervosa …).
Several decades of research have tempted to answer the question of the timing of the reactivation of GnRH secretion and the onset of puberty.As mentioned earlier, the hypothalamic-pituitary gonadal axis is functional during fetal/perinatal period, leading to the sexualization of external genitalia and specific regions of the nervous system.This activation is limited in time but offers a window of sensitivity to external factors such as endocrine disruptors (Parent et al., 2015;Hines et al., 2016).

Steroid-dependent mechanism
Early studies highlighted the role of the steroid negative feedback, the so-called "gonadostat" hypothesis (Frisch and Revelle, 1970).The "gonadostat" theory implies a higher sensitivity of GnRH neuronal network to the negative feedback of steroids: a steroiddependent mechanism.In the prepubertal period, GnRH secretion is less sensitive to the negative feedback of gonadal steroids, the GnRH pulse frequency increases leading to gonadotropin secretion and gonadal activation.In the sheep species, early post-natal gonadectomy leads to immediately increased levels of gonadotropins as in the postpubertal period.Replacing steroid gonadal hormones causes gonadotropins levels to go back to initial prepubertal values (Foster and Hileman, 2015).Similar findings were found in other mammals: hamster, ferret (Sisk and Foster, 2004).In heifers the negative feedback of estradiol declined as puberty approached (Day et al., 1987).However, in rat and rhesus monkey, the gonadostat theory is not sufficient to account for the low gonadotropins levels during infancy (Sisk and Foster, 2004).Interestingly, a steroid dependent mechanism exists at the end of the juvenile period of female rhesus monkey (Rapisarda et al., 1983).

Steroid independent mechanism
In rat and monkeys, after neonatal castration, gonadotropins levels remain low during the infantile period and increase progressively in the juvenile period to reach high levels as those expected at puberty.Such findings have been also reported in humans suffering from gonadal dysgenesis (Winter and Faiman, 2009).Although the precise neuronal target of gonadal steroid feedback is not clearly known, POA and the ArcN are involved in sensing estradiol negative feedback in gonadectomized prepubertal rats (Uenoyama et al., 2015).GnRH neurons, although located in the POA, are not considered as the primary target since they do not express the estrogen receptor α (ERα) albeit they do express ERβ (Hrabovszky et al., 2000;Herbison and Pape, 2001) but this later isoform does not seem to be involved in puberty onset.To account for this steroidindependent system, one assumption is that during infancy, inhibitory brain circuits block GnRH secretion; this break is released at puberty concomitantly with the onset of stimulatory brain circuits.GABA (γaminobutyiric acid) neurons are involved in the inhibition of GnRH neurons during juvenile period in several species.In the rhesus monkey, Terasawa's group showed the existence of a GABAergic break on gonadotropin secretion during the juvenile period.GABA level in the pituitary stalk (PS) and ME of juvenile monkeys is high but decreases during the peripubertal period (Mitsushima et al., 1994;Terasawa, 2005).The local infusion of GABA-A receptor antagonist bicuculline in the PS-ME of juvenile female rhesus monkey induces a rise in gonadotropins levels and the onset of ovarian cyclicity (Keen et al., 2011).The infusion of anti-sens mRNA encoding GAD67 (glutamic acid decarboxylase 67), a key enzyme for the synthesis of GABA, in juvenile rhesus monkey females triggered puberty onset with estrus cyclicity and ovulation (Kasuya et al., 1999;Fig. 3).
Both mechanisms co-exist to a different degree according to the species considered and also to the sex.One theoretical hypothesis would be that the steroidindependent mechanism provides a coarse regulation and will program the year (month) of puberty onset and the steroid-dependent mechanism will program the week/day when the first ovulation occurs.

Excitatory mechanisms
The pubertal reduction in GABAergic inhibition is accompanied by an increase in glutamate levels in the PS-ME, as well as an increase in the levels of the stimulatory neurotransmitters such as noradrenaline and Neuropeptide Y (NPY; Gore and Terasawa, 1991).

Neuropeptide Y
NPY is an appetite-stimulating neuropeptide and a neuromodulator of neuroendocrine functions.The interactions between NPY and neuroendocrine networks are complex and depend upon the sex and steroid environments.For example NPY is a potent stimulator of LH secretion in sex-steroid primed rats (Allen et al., 1985), whereas its intra-cerebroventricular (ICV) administration in gonadectomized rats inhibits LH release (McDonald et al., 1989).In the male Rhesus monkey, NPY exerts a negative effect on the GnRH pulse generator in prepubertal animals (Majdoubi et al., 2000).However in the female Rhesus monkey NPY release in the ME increases and is responsible for the observed increase in LH secretion at puberty onset (Gore et al., 1993).Two populations of NPY containing neurons have been described in the ArcN and the authors suggest that these two populations have distinct roles during the prepubertal period and at puberty onset (Majdoubi et al., 2000;Fig. 3).In the prepubertal ewe, NPY stimulates the expression of Lhb (β-LH subunit) in gonadotrope cells (Wańkowska and Polkowska, 2009).Neuroanatomical studies in prepubertal ewes demonstrate the presence of NPY inputs on Kp neurons in the ArcN (Polkowska et al., 2014) and on GnRH neurons in the POA (Norgren and Lehman, 1989;Tillet et al., 1989), thus suggesting two distinct pathways that can be involved in the stimulatory effect of NPY.The existence of 5 NPY receptors subtypes coupled to various signaling pathways and the existence of different hypothalamic and pituitary targets, can account for such opposite effects observed according to the sex, the steroid environment and the physiological state (Pralong, 2010).

Glutamate/NMDA
The excitatory amino acid glutamate and especially its NMDA subtype receptor are important components of the neural system that regulates sexual maturation.Multiple daily injections of NMDA agonists to immature rats (Smyth and Wilkinson, 1994) and monkeys (Urbanski and Ojeda, 1990) induce precocious puberty.On the contrary, administration of the noncompetitive NMDA antagonist, MK801, delays puberty onset (Veneroni et al., 1990).GnRH neurons receive direct glutamatergic inputs and express NMDA and kainite receptors (Fig. 3).Hypothalamic glutamate contents increase during the prepubertal period and reach maximal values at puberty onset.Glutamate receptors are ubiquitous in the CNS and they play important roles in many processes involving excitatory mechanisms, whether this increase in glutamatergic signaling is specific to puberty onset or whether it's a more general developmental process is not known (Parent et al., 2005).

Kisspetin/GPR54
In 2004, a new key component was discovered, originally named metastin due to its anti-mitotic properties and now named Kisspeptin (Kp; Matsui et al., 2004;Seminara, 2005).Kiss1 encodes a 54 aminoacids peptide Kp-54 (Kisspeptin-54) that cleaves into several shorter forms (Kp14, Kp13 and Kp10) forming the Kp family.Kp neurons strongly regulate the activity of GnRH neurons.Kp acts through a G-protein coupled receptor (GPCR): GPR54.Kp neurons are found in two distinct populations: ArcN and anteroventral periventricular nucleus (AVPV; Fig. 3).GnRH neurons express GPR54 and Kp fibers contact GnRH terminals in the ME.In humans, mutations in the GPR54 gene lead to an hyponadotropic hypogonadism (HH) characterized by a deficiency in pituitary secretion of gonadotropins which results in the impairment of pubertal maturation and of reproductive function (de Roux et al., 2003).Genetic models in rodents highlighted the central role of Kp/GPR54 system in the onset of puberty (Colledge and de Tassigny, 2009; Fig. 3).The Kp/GPR54 system is strongly regulated by metabolic factors and environmental factors, and could represent the central hub for decoding metabolic and environmental cues.

Glial regulation
When speaking of neuroendocrine regulations, most scientists focus on the roles played by neuronal circuits, neurotransmitters and neuromodulators and their cognate receptors.During the prepubertal period, although neuronal networks synaptically-connected to GnRH neurons govern the increase in GnRH secretion; glial cells contribute to the processes engaged through several mechanisms.Glial is a generic adjective to characterize several cell populations that are associated with GnRH neurons: astrocytes, tanycytes and olfactory unsheathing cells.For the sake of simplicity, we use the generic term, bearing in mind that different phenotypic cell types support it.One mechanism involves the production of growth factors acting on serine/threonine kinase receptors.Growth factors such as Transforming Growth Factor α (TGFα) and neuregulins acting on erbB receptors play a major role in glia-GnRH neurons communication.Activation of erbB receptors in glial cells associated with GnRH neurons, leads to the release of prostaglandin E2 (PGE2), which stimulates the electrical activity of GnRH neurons and the GnRH release (Prevot et al., 2003a(Prevot et al., , b, 2005;;Ojeda et al., 2008).Other growth factors such as TGFβ, IGF1, bFGF are secreted by glial cells and regulate directly the activity of GnRH neurons (Ojeda et al., 2010;Fig. 3).Besides the secretion of growth factors, glial cells release small molecules such as calcium, glutamate and ATP that affect the GnRH neuronal activity.Glial cells can also uptake K + ions and glutamate that accumulate in the extracellular space during neuronal activity through glial specific dedicated transporters.These mechanisms are of major importance in regulating neuronal electrical activity and excitability.These mechanisms of regulation are tightly dependent upon the distance between the membrane of the glial and the synaptic cleft (Giaume et al., 2010).
Another mechanism that can affect glia-GnRH neurons interactions is the modulation of adhesiveness of glial cells onto GnRH neurons.Glial cells interact with GnRH neurons via homophilic interactions involving Neural Cell Adhesion Molecule (NCAM) and synaptic cell adhesion molecule (SynCAM1).In contrast, the poly-syalilated form of NCAM, PSA-NCAM, prevents hemophilic interactions between adjacent glial and GnRH neuronal cells.Heterophilic interactions also exist via the neuronal membrane protein contactin and the glial receptor like protein tyrosine phosphatase-b (Parent et al., 2007).These cellto-cell interactions can trigger intracellular signaling cascades that can affect both glial and neuronal activities (Viguie et al., 2001;Parkash and Kaur, 2007;Sharif et al., 2013).Altering cell-to-cell communication through glial gap junctions or hemichannels decreases dramatically GnRH neuronal activity and GnRH secretion in vitro (Pinet-Charvet et al., 2015).Gapjunctions have previously been reported in the hypothalamus, particularly in the ArcN of female rats, where they are regulated by estrogen (Perez et al., 1990).Hypothalamic tanycytes, particularly the β-type which is closely associated with GnRH nerve terminals in the ME, express functional connexin-43 (Cx-43) hemichannels encoded by Gja1, which play a role in a glucose-sensing mechanism by releasing ATP (Orellana et al., 2012).The Gja1 (Cx-43) promoting region contains AP1 and AP2 sites and a series of half palindromic estrogen response elements suggesting that Cx-43 (Gja1) expression can be directly regulated by estrogen levels (Yu et al., 1994).Taken altogether, these studies suggest that glial cells might exert a control of GnRH neuronal network as important as the classical transynaptic model.
Therefore, several layers of neuronal and glial components are involved in controlling the onset of puberty, increasing the complexity of the system.The most important question remains: what determines the timing of the inhibitory break removal and/or the timing on excitatory inputs onset?

Puberty: environmental cues
The timing of puberty is maybe the best example of the interaction between genotype and environment.Puberty is a physiological event integrating several important central regulations at the crossroad of adaptation to environment: reproductive axis, feeding behaviour and nutritional controls, growth, seasonal rhythm, corticotropic axis and stress.

Nutrition and metabolism
Nutritional factors have been considered for a long time as the key factor in puberty onset.In humans, until the mid-20th century, a gradual decline in age at menarche (first menstruation) has been reported in most industrialized populations.It is generally admitted that this trend was due to gradual improvements in nutrition and healthcare (Sørensen et al., 2012) giving birth in the 70ies to the critical fat mass hypothesis according which, for a given species a critical fat mass is necessary for puberty onset.The link between nutrition and puberty onset was confirmed in numerous studies on laboratory animals, and also in farm animals.Adequate growth and adiposity are critical for the onset of puberty in mammals.Food restriction (Foster and Olster, 1984;Suttie et al., 1991) and excessive exercise during the juvenile period delay the onset of puberty (Manning andBronson, 1989, 1991).The mechanism involved is the maintenance of the juvenile high sensitivity to the negative feedback sensitivity to gonadal steroids.In contrast, increased adiposity advance the onset of puberty (Kaplowitz et al., 2001;Rosales Nieto et al., 2014).This occurrence is associated with attenuation of estradiol negative feedback and increased pulsatile release of LH (Gasser, 2006).Therefore, nutritional cues interact with gonadal steroid feedback to time the onset of puberty in females.These findings led to the concept of nutritional programming of puberty in cattle.Age at puberty in cattle is indeed influenced by food intake, food composition and body weight (BW).It is usually admit that puberty occurs at 55-65% of adult BW, depending on the breed considered (Freetly et al., 2011).However, the cost of supplemental feeding to reach this target BW earlier is not always compensated by a sufficient improve in reproduction and calf production (Davis Rincker et al., 2011).The permissive nutritional signals for puberty onset are metabolic cues such as glucose, insulin and leptin for the most studied factors.These metabolic markers signal the brain that the somatic growth and energy stores are sufficient to sustain pregnancy and lactation without threating the mother and foetus' health.Interestingly, these factors are also important for males, although the mechanisms involved may differ.Since the discovery of the fat-signalling hormone leptin (Zhang et al., 1994), whose blood level is proportional to the amount of adipose tissue (Frederich et al., 1995), a great amount of research work has tried to demonstrate that leptin is a hormonal messenger signalling the metabolic state for initiating puberty and also for fertility.Studies performed in rodents suggested that leptin administration could advance the onset of female puberty (Ahima et al., 1997).Humans with leptin deficiency due to mutations in the leptin gene or in the leptin receptor, and mouse models with inactivated leptin gene or leptin receptor gene, are obese and do not undergo puberty (Chehab et al., 1996).However leptin administration in healthy juveniles does not advance puberty onset.In ewes (Henry et al., 2011) and cows (Amstalden et al., 2002) leptin administration does not affect the secretion of LH but leptin prevents fasting-induced reduction in LH pulsatility in prepuberal heifers (Maciel, 2004).In addition to leptin, other hormones such as insulin, or nutrients such as glucose, fatty acids and amino-acids have been shown to regulate GnRH neuronal activity in a direct manner or via a complex glial/neuronal network.Among the critical neuronal pathways, hypothalamic NPY/agouti-related protein (AgRP) and proopiomelanocortin (POMC) neurons located in the ArcN are considered as the two major pathways mediating nutritional cues.Theses neurons express the leptin receptor and target GnRH neurons, setting the physical pathway for the control of puberty onset.A small subpopulation of Kp neurons in the ArcN express LepR (Louis et al., 2011) and may constitute another target for nutritional regulation see Sánchez-Garrido and Tena-Sempere (2013) for a review.However selective ablation of LepR in Kiss1 expressing neurons does not alter puberty onset and fertility (Donato et al., 2011).
Taken altogether, these studies support a permissive role of leptin in the metabolic gating of pubertal maturation (Barash et al., 1996;Cheung et al., 1997).

Photoperiod
In photoperiodic species, puberty onset will depend on the timing of the birth.For example in the ovine species lambs born at the end of the winter or during spring time reach puberty at the next breeding season in autumn, a younger age that those born during autumn, reaching puberty at the following breeding season 10-12 months later.This delay in puberty in autumn-born ewe lambs is due to a prolonged hypersensitivity to the negative steroid feedback (Foster and Hileman, 2015).Similar findings were observed for photoperiodic short-lived animals such as Siberian hamsters where spring born individuals mature rapidly and breed during the summer whereas young born in lid to late summer have a delayed puberty the next spring (Butler et al., 2007).Exposing Holstein heifers to long day photoperiod enhance BW gain and hasten the onset of puberty (Rius et al., 2005), a result that has been observed also for the seasonal Murrah buffalo species (Roy et al., 2016).In photoperiodic species, the variation in food intake and metabolism is an adaptive physiological mechanism allowing the storage of energy resources in anticipation of the harsh days of winter.The immune response is also sensitive to photoperiod, short days photoperiod enhance immunological defenses.This seasonal plasticity of the immune system is highly conserved and is in opposite phase with the breeding season, one explanation would be that the energy cost of both activating reproduction and maintaining the immune function at its higher level is too high (Walton et al., 2011).In dairy cows, short days photoperiod improve mammary gland capacity, prolactin secretion and immune function (Dahl, 2008).

Stress and corticotropic axis
Prolonged or chronic stress results in the suppression of gonadotropin secretion and the inhibition of reproduction.Acute stress has variable effects (Tilbrook et al., 2000).Studies on adaptive response processes highlighted a positive link between childhood adversities with accelerated female reproductive development.Longer-term health costs are traded off for increased probability of reproducing before dying Anim.Reprod., v.13, n.3, p.313-333, Jul./Sept.2016 via a process of accelerated reproductive maturation.Early adversity, early sexual maturation form the core component linking stress physiology with poor health later in life (Hochberg and Belsky, 2013).

Puberty: genetic determinants
While the timing of pubertal onset varies within and between different populations, it is a highly heritable trait, suggesting strong genetic determinants.Previous epidemiological studies estimate that 60-80% of the variation in pubertal onset is under genetic regulation (Parent et al., 2003;Gajdos et al., 2010).Abnormal pubertal timing affects up to 5% of adolescents and is associated with adverse health and psychosocial outcomes.
Oligogenic and complex genetic environmental interactions have now been identified, with physiological and environmental factors interacting in genetically susceptible individuals to alter their reproductive capacities.

Genetic factors associated with precocious puberty in Humans
Human precocious puberty is defined as the development of secondary sexual characteristics and elevated sexual hormones before 8 years of age in girls and 9 years of age in boys.There are two major forms of premature sexual maturation: inappropriate early activation of HPG axis that induces central precocious puberty (CPP) and peripheral precocious puberty (PPP) due to the increase of sex steroids with no activation of the HPG axis.Precocious puberty is highly deleterious since it will cause short stature, psychosocial problems and increase the risk of adulthood diseases.
Mutations in the LHCGR gene coding the LH receptor (LH-R) and leading to constitutive activation of the LH-R without ligand were the first mutations characterized in various family cases of peripheral precocious puberty limited to the male (Layman, 1999).These mutations affected only the male offspring and were without effect on the females.Recently cases of central precocious puberty have been associated with genetic variants affecting Kp signalling: mutation in the KISS1 gene encoding Kp (Silveira et al., 2010;Mazaheri et al., 2015) or activating mutation of the KISS1R gene encoding GPR54 the Kp receptor (Teles et al., 2008;Silveira et al., 2010;Fig. 4).One of these mutations was present at heterozygous state in patient's mother and grandmother suggesting incomplete sexdependent penetrance.Another possibility is that other genes could be involved in this phenotype evoking the oligogenicity concept in central precocious puberty as was well described for IHH (Mitchell et al., 2011).
Other cases of central precocious puberty are associated with mutations in the imprinted MKRN3 gene encoding the makorin ring finger protein 3, a gene located in the imprinted Prader Willi syndrome region (Settas et al., 2014;Simon et al., 2015;Fig. 4).Data from Human cases and animal models suggests that MKRN3 plays an inhibitory role in the reproductive axis and may represent a new pathway in pubertal regulation (Ong et al., 2009;Simon et al., 2015).MKNR3 is expressed ubiquitously.
Before 2000, clinical studies were individual case studies but now with the improvement of the methods of sequencing of the genome, the increase of the capacities of calculation and the improvement of the algorithms, the studies of association of genomic data allow to find genetic variants associated to the age in the puberty.With this process, more than 100 loci involved in the susceptibility to precocious puberty have been discovered.Among them the LIN28B locus is one of the most significant (Ong et al., 2009;Elks et al., 2010;Fig. 4).LIN28B is a human homolog of lin28 of Caenorhabtidis elegans, which was originally identified as a heterochronic regulator of developmental timing (Ambros and Horvitz, 1984) Deleterious mutations in lin28 resulted in precocious larval to adult development and a partial transformation in sexual phenotype (Ambros, 2011).The Lin28 proteins are potent and specific posttranscriptional repressors of the biogenesis of let-7 miRNAs, which are time-specific expressed miRNAs that control developmental timing (Zhu et al., 2010).
A recent meta-analysis suggests that the variant allele carriers, especially people with heterozygote genotype for ESR1 XbaI polymorphism and the wild allele for ESR1 PvuII polymorphism, are associated with precocious puberty susceptibility (Luo et al., 2015;Fig. 4).

Genetic factors associated with age at puberty in cattle
Age at first calving usually varied between 24 and 36 months, according to cattle breeds and is considered a key factor in terms of profitability and efficiency in both dairy and beef cattle.Likewise, bull puberty also shows significant differences within and among breeds.In dairy cattle, age at first has continually decreased during the last decades.
Improvement in nutrition and health have certainly contributed to an improve BW gain, but genetic selection for improved breeding and economic efficiency may also have indirectly impacted the onset of puberty (precocity; Mourits et al., 2000).Indeed, comparison of performances of 1970s and 1990s heifers from the same breed in New Zealand showed that modern heifers reached puberty at an earlier age than their predecessors, with a higher body weight than 20 years ago, meaning that mature size is different (Macdonald et al., 2007).As first calving at 24 months of age is becoming a common and general goal, one can safely assume that first-calving age will continue to decrease in the short term (Le Cozler et al., 2008).
Despite its economic importance, only a few studies have been conducted to identify genes and mutations associated with onset of puberty in either bulls or heifers.Most of these studies were done in beef cattle (mainly Angus), tropical breeds such as Brahman and Nelore cattle (Bos indicus cattle) and crosses which are reportedly older at puberty when compared with most Bos taurus breeds (Lunstra and Cundiff, 2003).Several parameters have been measured as a phenotype to study heifer puberty, from simple traits such as age at first service, age at first calving and age at first oestrus to more expensive and difficult to measure ones such as age at first corpus luteum (ultrasonography) or plasma progesterone concentration.For males, scrotal circumference, sperm quality (concentration, motility and morphology) as well as LH or IGF-1 circulating blood concentration have been monitored.One has to be aware that the nature of the quantitative puberty traits thus differs between studies.Moreover, their physiological meaning might be different than the strictly defined puberty onset.For example age at first oestrus does not mean age at puberty onset since oestrus behaviour is usually not present before the third oestrus cycle.Age at first calving is not age at first oestrus since the genital tract need several oestrus cycles to be fully developed in order to insure a full-length pregnancy.
Moderate to high heritability has been computed for heifer's age at puberty (0.2 to 0.48) and scrotal circumference (0.22 to 0.42; Vargas et al., 1998), suggesting that timing of puberty is likely to be a multigenic trait.Genetic correlations have also been observed between scrotal circumference or male IGF-1 blood concentration and heifer's age at puberty, suggesting that some common pathways may be involved in the two genders (Martinez-Velazquez and Gregory, 2003;Morris et al., 2010;Johnston et al., 2013).
Despite the multigenic nature of puberty onset, some major key player genes have been identified in humans, stimulating association studies in cattle, focused on some candidate genes.Polymorphisms in GNRHR, LHR and IGF were search for association with age of puberty in Angus male cattle (Lirón et al., 2012), showing significant association with one SNP located in IGF1.Likewise, polymorphisms in the LHR, FSHR and GNRHR were analysed in the Nellore breed, showing only association between FSHR and early puberty phenotype (Milazzotto et al., 2008).Furthermore, seven genes from the IGF1 pathway (IGF1R, IGFBP2, IGFBP4, EIF2AK3, PIK3R1, GSK3B and IRS1) were shown to be associated with heifer puberty in both Tropical Composite or Brahman breeds (Fortes et al., 2013).These findings support the hypothesis that IGF1 regulates arrival to puberty in male calves and also impact heifer puberty.In contrast to human and mouse, there are no evidences that genetic variation within GNRH, LH and its receptors could impact the regulation of pubertal timing in cattle.Based on their known effect on sexual precocity in mammals, 57 candidate genes related to lipid metabolism were also studied on a large panel of 1689 precocious and non-precocious Nellore heifers.Statistical analysis revealed that SNPs located within the FABP4 and PPP3CA gene had a significant effect on sexual precocity (Dias et al., 2015).
Genome-wide association studies (GWAS) using microsatellites or SNPs have also been set up to identify QTL regions and highlight to new candidate genes.A search for markers associated with heifer's age at puberty and age at first calving in the Animal QTLdb (Hu et al., 2016) retrieves about 350 markers located within roughly 200 QTL regions, irrespective to breeds.Likewise, 10650 makers within 60 regions have been associated with male puberty, mainly on the X chromosome.Several candidate genes have been proposed starting from these regions and regulatory networks have been constructed (Fortes et al., 2010a(Fortes et al., , b, 2011(Fortes et al., , 2016)).These findings suggest an enrichment of genes involved in axon guidance, cell adhesion, ErbB signaling, and glutamate activity, pathways that are known to affect pulsatile release of GnRH, which is necessary for the onset of puberty.In addition several TF were proposed as regulator of heifer's puberty, including ESRRG, PPARG, HIVEP3, TOX, EYA1, NCOA2, and ZFHX4.Combining GWAS and expression analysis in a multi-tissue omics also identified several key transcriptional regulators such as PITX2, FOXA1, DACH2, PROP1, SIX6 … (Canovas et al., 2014).U6 spliceosomal RNA was also proposed as a positional candidate gene associated with age at first calving (Nascimento et al., 2016).
Interestingly, only a few common genes can be identified between genes located within QTL associated with either heifer's or bull puberty and genes already known in human to be involved in puberty onset: HDAC8 and NR0B1 may play a role in male puberty, whereas CHST8, GABRA1, LEP and PROP1 may influence female puberty (Fig. 5).This finding suggests that cattle could provide new insight into the genetic basis of puberty in mammals.Consistent with the hypothesis of common pathways between genders, 16 common genes can be identified within heifer and bull QTL regions: ARL2, CAPN1, CDC42EP2, DPF2, FRMD8, MRPL49, PARPBP, POLA2, SAC3D1, Slc22a20, SNX15, SPDYC, TIGD3 , TM7SF2 , VPS51, ZFPL1.
Figure 5.Only a few genes known to be associated with puberty onset in human are also located within cattle QTL regions.Cattle QTL regions were identified using the Animal QTLdb, taking into account "Age at first calving" for females and "Scrotal circumference" for males.Regions associated with "Age at puberty" were spread over female or male according to the experiment.QTL regions were defined as the critical mapping interval for linkage studies or a 500kb interval centered on the most significant marker for GWAS studies.Ensembl database was used to list genes located within these intervals and OMIM was used to establish a list of genes associated with puberty in Human.The Venn diagram presents the number of common genes between these lists, showing a limited number of common QTL and genes involved in bull's and heifer's puberty and only a few human candidate genes located within cattle QTL regions.

How to get good oocytes at younger age?
The overall goal of a replacement heifer program is to rear heifers to reach a desired age and body weight early so that they initiate puberty, establish pregnancy, and calve easily at a minimal cost.In addition to the investment needed to raise heifers from birth to calving, heifers that calve earlier spend a greater proportion of their life producing milk, and therefore returning profit to a dairy, whereas heifers that calve later spend more time in a non-productive period before initiation of lactation.The development of replacement heifers is a major economic investment for all beef and dairy operations.The costs associated with heifer development cannot be recovered if heifers do not conceive and remain productive in the herd; therefore, heifers need to conceive early in the breeding season or risk being culled.Breeders can use various levers to meet these objectives.

Advancing puberty
Feeding and photoperiod (ovine species) were the two main levers used by farmers to advance puberty.Young juvenile heifers fed with high-concentrate diet have a better weight gain and an advanced puberty onset compared to control heifers.The timing of this nutritional support is important, there is a developmental window during the early juvenile period (between 4-6.5 months) during which, high-concentrate diet will be effective on the timing of puberty onset.Feed restriction after this point will have little effect on the timing of puberty (Cardoso et al., 2015).One could imagine that the qualitative nutritional value and the timing of nutritional programming are of importance and should benefit from a research effort in this field.
Although this is not recommended by Europe, hormonal treatment can be used to advance puberty onset.Hormonal treatments are efficient to advance the first ovulation when administrated during the late juvenile period (8-10 months) in pre-pubertal heifers.They involve the administration of GnRH agonists or hCG (human chorionic gonatodotropin).The GnRH agonist Buserelin acetate is commonly used for oestrus synchronization or for treating post-partum anoestrus in adult females.Continuous infusion of GnRH or GnRH agonist (Deslorelin) using sub-cutaneaous implants or minipumps to 8-10 months' old heifers stimulate LH secretion and induce ovulation 30-48 h after the placement of the implant (Dodson et al., 1990;Grasselli et al., 1993).However luteinisation and the production of progesterone are not consistently observed and this may cause short luteal phases.The continuous exposure to GnRH or GnRH agonists induces the desensitization of the GnRH-R signalling.After GnRH agonist implants removal, the animals do not respond to exogenous GnRH treatment for 12 days (Bergfeld et al., 1996).For these reasons, hCG is usually preferred.hCG will mimic the effect of endogenous LH surge and stimulate the ovulation of the dominant follicle.The luteotropic effect of hCG guarantees the formation of a functional corpus luteum and will have a beneficial effect on the initiation of pregnancy.Its major side effect is that hCG is a human hormone and as such its repeated administration causes the development of an acquired immunity that impedes future treatments to be efficient (De Rensis et al., 2010;Dahlen et al., 2011).Both GnRH agonistsand hCG-based treatments rely on peptidic or proteic Anim.Reprod., v.13, n.3, p.313-333, Jul./Sept.2016 substances that are not an environmental issue.In contrast to oestradiol-and progesterone-based hormonal treatments that have been used in the past in Europe or are still in use on the American, Asian and Australian continents.It would be interesting to test for Kp long life agonists that have been developed for the ovine species to see whether they could offer a more physiological activation of the central GnRH controlling system and thus avoiding the desensitization of GnRH-R signalling (Beltramo et al., 2015).

Collecting prepubertal oocytes
Another strategy is to overcome these problems by using in vitro production techniques and oocytes collection by Ovum Pick-Up (OPU) techniques.Despite the fact that large follicles are present before puberty, that good quality oocytes evaluated by the presence of compact cumulus can be collected by OPU, that the proportion of cleavages up to 8 cells after in vitro fertilization is correct, the rate of blastocysts obtained is low and their ability to produce successful pregnancy after embryo transfer is poor in comparison to data obtained from adult oocytes (Armstrong et al., 1992;Levesque and Sirard, 1994;Majerus et al., 1999;Landry et al., 2016).Ovarian stimulation using FSH can improve the rate of blastocyst formation, underlining the importance of hormonal environment to insure the oocyte competency to sustain development (Khatir et al., 1996).Different factors have been studied and sustain the cytoplasmic immaturity of prepubertal oocytes (Gandolfi et al., 1998;Oropeza et al., 2004;Bernal-Ulloa et al., 2016).Gene expression in blastocyst embryos relies mostly on post-transcriptional control of maternal transcripts accumulated during oocyte maturation.In calf oocytes, the expression of maternal transcripts differs from that of adult oocytes.Transcripts of PRDX2 and PRDX1 genes are in less quantities in oocytes collected from prepubertal animals in comparison to adult animals (Romar et al., 2011).

Conclusions
With the introduction of genomic selection 15 years ago, international agricultural politics have started to modify selection strategies, which now include puberty traits in order to advance puberty onset with the objective of reducing generation intervals.The selective pressure on onset of puberty will undoubtedly increase in a near future.Indeed, advances in molecular genetics have now made it possible to predict the total genetic value of animals by using genome-wide dense marker maps leading to the forthcoming of Genomic Selection (GS; Humblot et al., 2010).GS is of particular interest in cattle since the generation interval is long, artificial insemination bulls should be tested on their progeny before dissemination and some important traits such as fertility have a low heritability, due probably to a great sensitivity to environmental factors.Yearling bulls that have genomic breeding values information but lack phenotypic data on their daughters are often referred to as "genomic bulls".There has been an immense shift among the AI companies toward the use of genomic bulls in the past 3 years.Some AI companies use almost all genomic bulls as sires of sons, whereas other companies use a combination of genomic bulls and progeny-tested bulls (Schefers and Weigel, 2012).Instead of waiting a minimum of 4.5 years to use progeny-tested bulls as sires of sons, AI companies could now use the best DNA-tested young bulls by roughly 1 year of age.Due to economical constrains, AI companies are now looking for animals having an advancement of their puberty.
It's therefore of major importance to understand the link between these phenotypic changes, genetic determinants and environment.Indeed, GS de facto reduces the interval of generation and will speed up the selection process.This could be a great opportunity but may also increase the risk of disseminating unsuitable traits by lack of knowledge of their related pathways.Therefore, before implementing GS for QTL associated with puberty traits, it's crucial to evaluate whether or not this selection process may affect other reproductive characteristics or reduce the robustness and increase vulnerability to environmental changes.There is clearly a need for basic research on factors that control puberty in order to improve heifer development and fertility (Perry, 2016) and address the question of robustness.

Figure 3 .
Figure3.Schematic representation of neuroendocrine circuits.GnRH neurons receive inputs from Kp neurons located in the AVPV and ArcN, NKB neurons located in the ArcN , endorphin neurons located in the ArcN , GABA and glutamate from interneurons (grey boxes = neurotransmitters, purple boxes = neuroanatomical structure).Kp neurons, NPY neurons and GABA neurons are sensitive to E2, leptin and insulin respectively (hormones = red boxes).Glial cells (astrocytes and tanycytes, orange pentagons) in the microenvironment of GnRH neurons can release glutamate, growth factors such as TGFβ1, bFGF that stimulates the activity of GnRH neurons.Glial cells can also uptake glutamate from extracellular space.Glial cells also release neuregulins (NRGs) and TGFα, stimulating the release of PGE2 by neighboring glial cells, which stimulates GnRH neurons.Glial cells also release Ca 2+ , ATP that regulate GnRH neurons' activity.

Figure 4 .
Figure 4. Genetic factors associated with pathological puberty delay or advance in humans.This figure summarizes how genetic factors associated with pathological conditions in humans are affecting cellular processes at hypothalamic and pituitary levels.From general cellular function GnRH neuron development and migration, GnRH synthesis, bioactivity and secretion, energy homeostasis, gonadotrope stimulation and endocrine feedbacks.