General principles of estrus regulation

During pro-estrus, when the corpus luteum (CL) is regressed and the concentration of (P4) is decreased, the dominant follicle, deviated from a cohort of antral follicles, matures under the influence of luteinizing hormone (LH) and follicle stimulating hormone (FSH; Allrich, Reference Allrich1994). FSH plays an important role at the beginning of follicular development, whereas LH is important for follicular growth up to ovulation (Ginther et al., Reference Ginther, Wiltbank, Fricke, Gibbons and Kot1996). The dominant follicle secretes an increasing amounts of estradiol (E2) during the development to preovulatory size (Allrich, Reference Allrich1994). E2 is involved in important neuroendocrine mechanisms regulating estrus. E2 inhibits gonadotropin releasing hormone (GnRH) secretion from the hypothalamus and LH secretion from the pituitary throughout most of the cycle. However, during pro-estrus, elevated E2 levels increase the secretion of GnRH, which together with direct effects of E2 on the pituitary, triggers the LH surge (Glidewell-Kenney et al., Reference Glidewell-Kenney, Hurley, Pfaff, Weiss, Levine and Jameson2007), which induces ovulation. Once an oocyte is successfully ovulated, the remains of the follicle form a new P4-producing CL. Progesterone maintains the readiness of the endometrium for receiving the embryo. If conception has failed, the CL regresses, P4 levels decrease and the cycle restarts.

Estrous behavior

The estrous cycle of cows lasts for approximately 21 days. The interval between onset of mounting behavior and ovulation in cows is approximately 27 h (Lopez et al., Reference Lopez, Bunch and Shipka2002; Roelofs et al., Reference Roelofs, van Eerdenburg, Soede and Kemp2005b). In modern Holstein cows the duration of estrus, defined as the time between first and last recorded standing event, has been reported to be 7 h (Dransfield et al., Reference Dransfield, Nebel, Pearson and Warnick1998; Lopez et al., Reference Lopez, Satter and Wiltbank2004). In contrast, Esslemont and Bryant (Reference Esslemont and Bryant1976) reported an average duration of estrus of 14.9 h in Friesian cattle in 1976. Table 1 summarizes different behavioral signs of estrus in cows. At the start of estrus, a cow typically sniffs the vulva of other cows and rests her chin on the back of others. Such behavior is followed by mounting of other cows and ultimately the cow displays standing heat (Roelofs et al., Reference Roelofs, van Eerdenburg, Soede and Kemp2005b). Van Eerdenburg et al. (Reference Van Eerdenburg, Loeffler and Van Vliet1996) defined a protocol based on these behavioral signs in order to detect whether a cow is in heat. As shown in Table 1, not all cows express all behavior traits. Lyimo et al. (Reference Lyimo, Nielen, Ouweltjes, Kruip and van Eerdenburg2000) and Roelofs et al. (Reference Roelofs, Bouwman, Dieleman, Van Eerdenburg, Kaal-Lansbergen, Soede and Kemp2004) showed that the highest behavioral score of cows in estrus, based on the estrous behavior signs given in Table 1, correlates positively with maximum plasma E2 concentrations, but no correlation was found between E2 levels and specific estrous behaviors (Cook et al., Reference Cook, Winters, Horstman and Allrich1986; Coe and Allrich, Reference Coe and Allrich1989). Because the percentage of cows displaying standing heat has declined over the last decades (reviewed by Dobson et al., Reference Dobson, Walker, Morris, Routly and Smith2008), it is more difficult to detect estrus based on standing heat. Therefore, other methods to detect and quantify estrus have been proposed by Roelofs et al. (Reference Roelofs, van Eerdenburg, Soede and Kemp2005a; pedometers) and Lovendahl and Chagunda (Reference Lovendahl and Chagunda2009; electronic activity tags). Little is known about the underlying mechanisms and the level of genetic control of specific estrous behaviors, but collection of quantifiable data could be helpful in the research of genetic mechanisms (Schutz and Pajor, Reference Schutz and Pajor2001).

Table 1

Behavioral signs of estrus in cowsFootnote *

* Adapted from Van Eerdenburg et al. (Reference Van Eerdenburg, Karthaus, Taverne, Merics and Szenci2002) and Roelofs et al. (Reference Roelofs, van Eerdenburg, Soede and Kemp2005b).

A ‘normal’ endocrinological cycle is prerequisite for estrus and estrous behavior. However, ovulation is not necessarily accompanied by estrous behavior (‘silent estrus’ (Allrich, Reference Allrich1994), indicating that physiological events and behavior are in part based on different mechanisms. In dairy cows, the first postpartum ovulation occurs often without clear signs of estrous behavior (Kyle et al., Reference Kyle, Callahan and Allrich1992). This ‘silent estrus’ is thought to be a result of high E2 concentrations from fetal origin at the end of gestation, which induces ‘refractoriness’ in the hypothalamus to E2 at the first postpartum ovulation. The CL produced after the first ovulation provides the P4 that removes this refractory state and facilitates the behavioral expression of the subsequent estrus (Allrich, Reference Allrich1994).

Endocrine regulation of estrous behavior: the central role of E2

E2 plays a key role in the regulation of endocrine and behavioral events associated with the estrous cycle. In many experiments that are performed to study the female reproduction, estrus is artificially induced by administering E2 (e.g. Fabre-Nys et al., Reference Fabre-Nys, Martin and Venier1993). E2 plays a central role in triggering the gonadotropin surge and ovulation as well as in facilitating the estrous behavior, and thus E2 indirectly synchronizes mating and ovulation. The patterns of GnRH synthesis and pulsatile release from the hypothalamus are mainly regulated by E2 (Smith and Jennes, Reference Smith and Jennes2001) and P4 (Richter et al., Reference Richter, Robinson, Lozano and Evans2005; Zalányi, Reference Zalányi2001). E2 stimulates LH synthesis, but at levels below a certain threshold value it inhibits the release of LH. Above this threshold, the inhibitory effect on LH release switches to a stimulatory effect (reviewed by Reinecke and Deuflhard, Reference Reinecke and Deuflhard2007), which results in the LH surge. The shift from inhibition to stimulation may be dependent on the site of action of E2, that is, a switch from membrane signaling to genomic signaling (Arreguin-Arevalo and Nett, Reference Arreguin-Arevalo and Nett2006). The LH surge is driven by an increased pituitary responsiveness to GnRH, which is determined by the amount of GnRH receptors (GnRH-Rs) expressed on gonadotropes. Pulsatile GnRH release, facilitated by high E2 concentrations during the preovulatory period, elevates GnRH-R gene expression, whereas high P4 concentrations in the luteal phase inhibits the GnRH-R gene expression (reviewed by Weiss et al., Reference Weiss, Polack, Treeck, Diedrich and Ortmann2006).

E2 plays a pivotal role in the induction of estrous behavior (Pfaff, Reference Pfaff2005). It has a self-amplifying effect as it stimulates the expression of estrogen receptors (ERs) in the brain, which is thoroughly investigated in rodents (Pfaff et al., Reference Pfaff, Kow, Loose and Flanagan-Cato2008). The duration of estrous behavior in sheep was found to depend mostly on the duration of E2 presence rather than on its maximum concentration (Fabre-Nys et al., Reference Fabre-Nys, Martin and Venier1993). The effects of E2 are highly similar in different species, although threshold concentrations for the induction of estrous behavior may vary between animal species, for example, 0.4 μg/kg in sheep and 10 μg/kg in rats (Fabre-Nys and Gelez, Reference Fabre-Nys and Gelez2007). In sheep, Saïd et al. (Reference Saïd, Lomet, Chesneau, Lardic, Canepa, Guillaume, Briant, Fabre-Nys and Caraty2007) demonstrated that estrous behavior required lower E2 concentrations than required for the LH surge and that estrous behavior can be induced independently of the LH surge. Estrous behavior and the LH surge cannot only be separated by experimental reduction of E2 levels, but also by stress (Dobson et al., Reference Dobson, Walker, Morris, Routly and Smith2008). Lameness, an example of a stress inducing condition, was found to reduce behavior score (based on signs given in Table 1) of cows in estrus (Walker et al., Reference Walker, Smith, Jones, Routly and Dobson2008) and to inhibit LH surge and ovulation (Dobson et al., Reference Dobson, Walker, Morris, Routly and Smith2008) whereas incidence of estrus was not reduced (Walker et al., Reference Walker, Smith, Jones, Routly and Dobson2008), which could result in lower pregnancy rates. These observations suggest that stress, caused by lameness, reduces P4 exposure before estrus (Walker et al., Reference Walker, Smith, Jones, Routly and Dobson2008) and/or E2 production by the dominant follicle and thereby reduces expression of estrous behavior (Dobson et al., Reference Dobson, Walker, Morris, Routly and Smith2008).

In cows and other domestic ruminants, the behavioral expression of estrus is preceded by a luteal phase of 12 to 15 days during which P4 concentrations are high (Fabre-Nys and Gelez, Reference Fabre-Nys and Gelez2007). High P4 concentrations during the luteal phase inhibit the E2-induced gonadotropin surge by reducing pituitary responsiveness to GnRH (Attardi et al., Reference Attardi, Scott, Pfaff and Fink2007; Richter et al., Reference Richter, Robinson, Lozano and Evans2005). The duration of P4 presence and the P4 amplitude in the luteal phase influence the time interval between rise in E2 levels and the induction of estrous behavior and the LH surge, probably by affecting the neural mechanisms that are involved in GnRH release (Skinner et al., Reference Skinner, Harris and Evans2000). The exact functions of P4 in the priming as well as the inhibition of estrous behavior are debated (Zalányi, Reference Zalányi2001; Weiss et al., Reference Weiss, Polack, Treeck, Diedrich and Ortmann2006; Attardi et al., Reference Attardi, Scott, Pfaff and Fink2007) and seem to differ between species (Fabre-Nys and Gelez, Reference Fabre-Nys and Gelez2007).

Metabolic disturbances

High milk production affects the energy metabolism, which can disturb the endocrine signaling (Roche, Reference Roche2006). Altered energy metabolism in high milk yielding cows can cause decreased levels of E2 and inhibit estrous behavior (Lopez et al., Reference Lopez, Satter and Wiltbank2004). Cows selected for high milk yield are genetically induced to a more negative energy balance (Veerkamp et al., Reference Veerkamp, Beerda and van der Lende2003) as they spend a relatively large proportion of the available nutrients on milk production, which can cause fertility problems during a period of negative energy balance (Chagas et al., Reference Chagas, Bass, Blache, Burke, Kay, Lindsay, Lucy, Martin, Meler, Rhodes, Roche, Thatcher and Webb2007). One possible route by which metabolic stress can inhibit estrous behavior is via insulin-like growth factor-1 (IGF-1). IGF-1 production is inhibited during negative energy balance. IGF-1 receptor signaling in the brain (Velazquez et al., Reference Velazquez, Spicer and Wathes2008) is needed for the positive effect of E2 on the release of LH and for normal E2 priming of estrous behavior (Etgen et al., Reference Etgen, González-Flores and Todd2006; Mendez et al., Reference Mendez, Wandosell and Garcia-Segura2006). Furthermore, concentrations of other metabolic factors that are known to affect dairy cow fertility, for example, insulin, leptin and growth hormone, interact with IGF-1 levels (Diskin et al., Reference Diskin, Mackey, Roche and Sreenan2003; Chagas et al., Reference Chagas, Bass, Blache, Burke, Kay, Lindsay, Lucy, Martin, Meler, Rhodes, Roche, Thatcher and Webb2007).

Changes in reproductive physiology that are associated with high milk production may in part be explained by elevated P4 and E2 clearance rates, as described in the physiological model of Wiltbank et al. (Reference Wiltbank, Lopez, Sartori, Sangsritavong and Gümen2006). In this model, clearance rates of hormones by the liver of a lactating cow are increased as a result of elevated feed intake, leading to an increased liver blood flow and metabolic activity. With a similar level of hormone production, circulating hormone levels would thus be lower. The model also provides an explanation for decreased duration of estrus: elevated E2 metabolism means a more rapid decrease in circulating E2 after the LH surge. Combining the facts that E2 is an important regulator of estrous behavior in cows (Lyimo et al., Reference Lyimo, Nielen, Ouweltjes, Kruip and van Eerdenburg2000) and that increased level of milk production is associated with decreased E2 concentrations (Lopez et al., Reference Lopez, Satter and Wiltbank2004), smaller follicular size (Diskin et al., Reference Diskin, Mackey, Roche and Sreenan2003) and shorter duration of estrus (Wiltbank et al., Reference Wiltbank, Lopez, Sartori, Sangsritavong and Gümen2006), it seems reasonable to conclude that lower E2 levels are (partly) responsible for the poor behavioral expression of estrus in modern dairy cows (Chagas et al., Reference Chagas, Bass, Blache, Burke, Kay, Lindsay, Lucy, Martin, Meler, Rhodes, Roche, Thatcher and Webb2007).

Estrogen signaling in the brain

Brain areas that are known to be involved in the regulation of female sexual behavior include the arcuate nucleus (ARC), ventromedial nucleus (VMN) and preoptic area (POA) of the hypothalamus (reviewed by Molenda-Figueira et al., Reference Molenda-Figueira, Williams, Griffin, Rutledge, Blaustein and Tetel2006). In addition to these areas of the hypothalamus, the hippocampus and amygdala are known to regulate the behavioral aspects of estrus. The amygdala (Zhou et al., Reference Zhou, Zhang, Cohen and Pandey2005) and hippocampus (Frye and Rhodes, Reference Frye and Rhodes2008) are involved in the reduction of anxiety and aggression, and in this way can facilitate sexual behaviors that result from generalized arousal of the brain. E2 and other hormones cause up- or downregulation in these brain areas of a number of genes that are believed to be involved in estrous behavior (Table 2). E2 increases the sensitivity of neurons for itself by inducing ER gene expression (Walf and Frye, Reference Walf and Frye2006 and Reference Walf and Frye2008). The E2-receptor complex acts as transcription factor that regulates the expression of a large number of genes (Molenda-Figueira et al., Reference Molenda-Figueira, Williams, Griffin, Rutledge, Blaustein and Tetel2006). Apart from genomic (classical ER) signaling, the estrogenic control of estrous behavior also involves membrane signaling mechanisms via secondary messengers like phosphoinositide 3 kinase, cAMP response element binding proteins and extracellular signal regulated kinases (Mendez et al., Reference Mendez, Wandosell and Garcia-Segura2006; Kelly and Rønnekleiv, Reference Kelly and Rønnekleiv2009; Micevych and Dominguez, Reference Micevych and Dominguez2009).

Table 2

Overview of above-mentioned genes involved in the regulation of estrus

ERα = estrogen receptor-α; ERβ = estrogen receptor-β; E2 = estradiol; P4 = progesterone; GnRH = gonadotropin releasing hormone; GnRH-R = gonadotropin releasing hormone receptor; PI3K = phosphoinositide 3 kinase; IGF-1 = insulin-like growth factor-1; LH = luteinizing hormone.

Arousal and lordosis behavior in rodents

Rodents are often used as a model to study the regulation of fertility in mammals, for example, using genomic approaches (e.g. Laissue et al., Reference Laissue, L’Hôte, Serres and Vaiman2009). The gene expression studies of Pfaff and coworkers revealed several mechanisms that are involved in arousal (Frohlich et al., Reference Frohlich, Ogawa, Morgan, Burton and Pfaff1999) and, more specific, lordosis in rodents (Kow and Pfaff, Reference Kow and Pfaff1998). Arousal, a general activation of brain and behavior, precedes the lordosis response and results from signaling by neurotransmitters like norepinephrine (Lee and Pfaff, Reference Lee and Pfaff2008). The expression in the brain of estrogen receptor-α (ERα) and its downstream effects are essential for arousal, as knockout of ERα reduced arousal responses in mice (Garey et al., Reference Garey, Goodwillie, Frohlich, Morgan, Gustafsson, Smithies, Korach, Ogawa and Pfaff2003; Mong et al., Reference Mong, Easton, Kow and Pfaff2003a). E2-induced down-regulation of prostaglandin-D synthase in the POA increases arousal response (Mong et al., Reference Mong, Devidze, Goodwillie and Pfaff2003b) and prostaglandin-D synthase downregulation is associated also with lordosis (Pfaff et al., Reference Pfaff, Kow, Loose and Flanagan-Cato2008). The initial step in the induction of lordosis is the E2 controlled alteration of neuronal activity in the VMN. Estrogen priming alters gene expression in VMN neurons, resulting in the activation of a variety of neurotransmitters and neuropeptides. For example, E2 induces expression of adrenergic receptor genes in the VMN (Lee et al., Reference Lee, Kyrozis, Chevaleyre, Kow, Devidze, Zhang, Etgen and Pfaff2008) and increases the proportion of neurons that respond to stimulation of adrenergic receptors, which is the first step of a signal transduction pathway resulting in lordosis behavior (reviewed by Lee and Pfaff, Reference Lee and Pfaff2008). Another example is the E2-induced expression of glial specific genes, including glutamine synthetase, in the ARC and VMN nuclei, and in the amygdala and hippocampus, thus facilitating the glutamatergic neurotransmission important for estrous behavior (Blutstein et al., Reference Blutstein, Choleris, Jasnow, Pfaff and Mong2006). At least nine genes, expressed in the rodent hypothalamus, are known to be turned on following the binding of estrogen to its receptor. In the VMN, binding of E2 to ERα activates the expression of genes for rRNA and growth, nNitric oxide synthase, adrenergic and muscarinic receptors, enkephalin and opioid receptors, P4 receptor, and oxytocin and oxytocin receptor (Table 2). In addition, binding of E2 to ERβ activates genes for P4 receptor, and oxytocin and oxytocin receptor. In the POA, E2 binding to ERα upregulates GnRH and GnRH-R genes and downregulates prostaglandin-D synthase (summarized by Pfaff et al., Reference Pfaff, Kow, Loose and Flanagan-Cato2008). Together, the products of these genes play a role in the induction of the behavioral expression of estrus. A recent study of Sica et al. (Reference Sica, Martini, Viglietti-Panzica and Panzica2009) in female mice showed changes in nNitric oxide synthase expression in the hypothalamus during the estrous cycle. Increased numbers of nNitric oxide synthase immunoreactive neurons were found in the ARC during proestrus and in the POA during estrus. As these regions show large numbers of ERα, this study supports the conclusion of Pfaff and coworkers that E2 modulates expression of nNitric oxide synthase, which stimulates estrous behavior via activation of the nitric oxide signaling pathway (Sica et al., Reference Sica, Martini, Viglietti-Panzica and Panzica2009). The estrogen-induced regulation of lordosis and synchronized ovulation in rodents can be described in five modules (Pfaff, Reference Pfaff2005). (i) Preceding estrus, E2 induces expression of genes involved in growth of dendrites and synapses of VMN neurons that are involved in facilitating sexual behavior. (ii) P4 administration after estrogen priming amplifies the effect of estrogen on reproductive behavior via upregulation of several transcripts. (iii) The presence of estrogens induces expression of several genes (examples are mentioned above) involved in behaviors that prepare the animal for mating. These genes establish analgesia, social recognition and reduction of anxiety and aggression. (iv) E2 induced upregulation of neurotransmitter receptors in VMN neurons primes the neural circuit that triggers the lordosis behavior. (v) E2 elevates GnRH, which stimulates the ovulatory gonadotropin release and facilitates estrous behavior. As E2, through its effects on GnRH and LH, also regulates the LH surge and ovulation, E2 indirectly synchronizes mating and ovulation. The lordosis reflex has been used as a behavioral model to study the functioning of serotonin (Uphouse, Reference Uphouse2000; Uphouse et al., Reference Uphouse, Hiegel, Perez and Guptarak2007) and E2 signaling (Micevych and Dominguez, Reference Micevych and Dominguez2009; Micevych et al., Reference Micevych, Kuo and Christensen2009). Although these studies do not aim directly to unravel the regulation of estrous behavior, they support the findings of Pfaff and coworkers that E2 induces estrous behavior via ER gene expression and membrane signaling.

Near to no research has been carried out on E2-induced gene expression related to induction of estrous behavior in cattle, but there are reasons to assume that the mechanisms are similar. Estrous behavior in ruminants, like cows and sheep, is controlled by E2 levels (Lyimo et al., Reference Lyimo, Nielen, Ouweltjes, Kruip and van Eerdenburg2000; Saïd et al., Reference Saïd, Lomet, Chesneau, Lardic, Canepa, Guillaume, Briant, Fabre-Nys and Caraty2007). In rodents, E2 reduces anxiety and therewith stimulates locomotion and exploratory behavior (Mong and Pfaff, Reference Mong and Pfaff2003c), and a similar E2-induced increase in activity is seen in cows (Roelofs et al., Reference Roelofs, van Eerdenburg, Soede and Kemp2005a). Another parallel that can be drawn between rodents and ruminants is that the brain areas shown to be highly involved in regulation of estrous behavior in rodents (ARC, VMN and POA) are also the regions with high concentrations of ER during estrus in ewes (Lehman et al., Reference Lehman, Ebling, Moenter and Karsch1993; Stormshak and Bishop, Reference Stormshak and Bishop2008). Most gene expression studies dealing with bovine fertility are focused on follicle development and changes in ovarian tissue (Zielak et al., Reference Zielak, Forde, Park, Doohan, Coussens, Smith, Ireland, Lonergan and Evans2007; Mihm et al., Reference Mihm, Baker, Ireland, Smith, Coussens, Evans and Ireland2006). Beerda et al. (Reference Beerda, Wyszynska-Koko, te Pas, de Wit and Veerkamp2008) compared gene expression profiles in brain samples of Holstein Friesian heifers in (pro-)estrus to those in heifers in luteal phase. Quantitative scores for estrous behavior are linked to gene expressions in the pituitary, hypothalamus, hippocampus, amygdala and ventral tegmental area (VTA). The first analyses of the VTA indicated that cyclical changes in the expression of genes regulating cell morphology and adhesion were linked to the appropriate expression of estrous behavior in dairy cows.