Characterization of Luteinizing Hormone and Luteinizing Hormone Receptor and Their Indispensable Role in the Ovulatory Process of the Medaka

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare’s serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.     

Involvement of cAMP-Dependent protein kinase and intracellular free calcium ions in regulation of ovulation of the follicle-enclosed oocytes ofRana temporaria by gonadotropic hormones

The inhibitor of cAMP-dependent protein kinase N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (H-8) (12.5–50 μM) decreased the rate of ovulation of the follicle-enclosedRana temporaria oocytes induced by the homologous pituitary extract in amphibian Ringer solution and in a chloride-free medium. The inhibitor of voltage-dependent calcium channels diltiazem (10 and 100 μM) decreased the rate of ovulation in Ringer solution but did not affect it in a chloride-free medium or decreased the ovulation inhibitory effect of this medium. It was concluded that cAMP-dependent protein kinase and intracellular free calcium ions were involved as second messengers in the gonadotropin regulation not only in maturation of amphibian oocytes but also in ovulation.     

Nicotine stimulates maturational gonadotropin (GtH2) release from carp (Cyprinus carpio L.) pituitary cells

1. Perifusion of dispersed pituitary cells and pituitary cell culture was used to investigate the effects of cholinergic drugs on the secretion of maturational gonadotropin (GtH₂) in carp.2. Nicotine strongly, and in a dose dependent manner, stimulated GtH₂ release in male and in female carp (from 10⁻⁸M in the Perifusion and 10⁻¹⁰M in the cells cultures).3. Nicotine is 10 times more active in females than in males.4. The results suggest that in carp, nicotine stimulates GtH₂ release directly from the pituitary cells, indicating a possible involvement of a cholinergic system in the regulation of GtH₂ secretion in teleost fish.     

Effects of pinealectomy on pituitary gonadotrophs, pituitary gonadotropin potency and hypothalamic gonadotropin releasing activity in Notemigonus crysoleucas

The effects of pinealectomy on pituitary gonadotrophs, pituitary gonadotropin potency and hypothalamic gonadotropin releasing activity were examined in the cyprinid teleost, Notemigonus crysoleucas, exposed to various photoperiod-temperature regimes. In fish exposed to a long photoperiod-warm temperature regime, pinealectomy resulted in a decrease in gonadal activity, in hypothalamic gonadotropin releasing activity and an increase in pituitary gonadotropin potency. Fewer gonadotrophs were present in the pituitary of sham operated fish than in the pituitary of pinealectomized fish. Ovarian development was more rapid in sham operated than in pinealectomized fish exposed to a long photoperiod–low temperature regime. Pituitary gonadotropin activity was also greater in shams than in pinealectomized fish. A short photoperiod-warm temperature regime retarded ovarian development in N. crysoleucas. Pinealectomy reversed this trend. Gonadotrophs made up a greater area of the pituitary in pinealectomized fish than in shams under these conditions. Gonadotropin potency of the pituitary and hypothalamic gonadotropin releasing activity were also greater in pinealectomized fish than in shams. The area of the pituitary occupied by gonadotrophs was greater in pinealectomized than in sham operated animals maintained on a short photoperiod-low temperature regime. Pituitary gonadotropin activity was also greater in pinealectomized fish as compared to shams. Pituitary gonadotropin potency varies diurnally in animals maintained on both short and long photoperiods; the rhythm of variation differs depending on photoperiod. Pinealectomy alters the diurnal rhythm of pituitary gonadotropin potency in animals exposed to both long and short photoperiods. It is concluded that pinealectomy has a pronounced effect on reproductive activity in N. crysoleucas. The effects of pinealectomy on reproduction vary with photoperiod, but are mediated via the hypothalamus and pituitary. In fish exposed to long daylengths the pineal favours reproductive activity, but the epiphysis retards reproductive processes in animals maintained on short photoperiods.     

Extra-pituitary action of gonadotropin releasing hormone: possible application

Chronic administration of a potent gonadotropin releasing hormone inhibits ovulation in women. The suppression of gonadal function during long term treatment with the GnRH analogues is ascribable to inhibition of gonadotropin secretion caused by the down regulatory action of the decapeptide at the pituitary level. Reduced progesterone production with premature onset of menstruation has been observed in women injected with the agonist during the midluteal phase. The decapeptide however, has no effect onin vitro human ovarian steroidogenesis. Specific receptors for GnRH have been located on rodent ovarian cells, but corpora lutea of rhesus monkey and human ovaries seem to lack these receptors. The luteolytic effect in women thus appears to be central in origin and not a direct effect on the corpus luteum. Recently, a superactive agonist of GnRH given around the peri-implantation period has been shown to terminate pregnancy in baboons. Monoclonal antibodies against GnRH administered during the same period in a fertile cycle also abrogated pregnancy in these animals. Using immuno-enzymatic techniques GnRH has been localized on the placenta. GnRH also exerts a stimulatory effect on hCG production by the placental villi maintained in culture. Addition of anti-luteinizing hormone releasing hormone antibodies blocks this effect completely. It seems that placenta is the only other tissue besides the pituitary where GnRH has probably a regulatory role in the human female.     

溴隐亭不同给药方案治疗高泌乳素血症女性性腺激素促排卵临床研究

目的:研究溴隐亭不同给药方案在治疗高泌乳素血症(HPRL)女性不育症中的临床疗效,关注其对女性促性腺激素诱导排卵的影响。方法:本研究共纳入60例就诊于我院的确诊为高泌乳素血症不孕不育患者,随机分为两组。分为研究组与对照组:研究组采取先口服溴隐亭调整血清泌乳素水平至正常后予以促性腺激素诱导排卵;对照组采取促性腺激素与溴隐亭同步治疗方案。结果:观察两组患者的促排卵周期数、平均用药天数、雌二醇水平及妊娠率,两组治疗前后的血清泌乳素都显著改善(P0.05);但是两组之间相比,采取溴隐亭药物治疗后诱导排卵的研究组在促排卵、雌二醇水平和妊娠率方面具有显著优势(P0.05)。结论:采用溴隐亭治疗高泌乳素血症患者,调整至正常后再使用促卵泡激素药物促排卵治疗不孕不育具为较优的治疗方案。     

Response of superfused goldfish pituitary fragments to mammalian and salmon gonadotropin-releasing hormones

Salmon and mammalian gonadotropin-releasing hormones (sGnRH, mGnRH) were tested for their ability to stimulate $\text{in vitro}$ gonadotropin (GtH) release from superfused goldfish pituitary fragments. A two minute exposure to either peptide was sufficient to stimulate a dose-dependent increase in GtH release which reached maximum levels in 15 minutes and returned to baseline within one hour. Both peptides were approximately equipotent in stimulating GtH release, as was a superactive analog of mGnRH. These results demonstrate that sGnRH is capable of directly stimulating GtH release from teleost pituitary tissue, and that structural differences between the three peptides tested do not result in significant differences in $\text{in vitro}$ bioactivity.     

Use of Carbopol resin for carp pituitary administration improves the fertilization percentage of northern pike (<Emphasis Type="Italic">Esox lucius</Emphasis> Linnaeus) eggs in commercial hatcheries

The efficiency of northern pike (Esox lucius Linnaeus) fry production via hormonal treatment of wintered broodstock is, in general, relatively low due to low egg fertilization percentage. It has been experimentally demonstrated that administration of acetone-dried carp pituitary extract in a slow-release vehicle of aqueous dispersion of Carbopol 971 P resin (CP) resulted in a higher mean fertilization percentage, possibly because the gradual hormone action could optimally control ovarian follicle maturation and ovulation. This new method of hormonal induction of ovulation was tested in a large-scale hatchery production of northern pike fry. In the present study, data on fertilization percentage collected between 2002 and 2006 at two prominent hatcheries in Hungary were analyzed. Administration of acetone-dried carp pituitary in a 2.5% CP vehicle resulted in higher fertilization (76.4 ± 7.7%; mean ± SD) compared to the saline vehicle group (59.3 ± 10.8%). The vehicle used did not affect the ovulation ratio. According to these results from large-scale production, the new method could be suitable to have wide application for induced breeding of northern pike. Guest editors: J. M. Farrell, C. Skov, M. Mingelbier, T. Margenau & J. E. Cooper International Pike Symposium: Merging Knowledge of Ecology, Biology, and Management for a Circumpolar Species     

Hypertrophic changes in the ovarian chromaffin tissue (catechol storing tissue) under the influence of the pituitary and its theoretical implications in the teleost fish, Nandus nandus.

Evidence is presented in this work for the existence of a special chromaffin tissue in the ovary of the percoid teleost fish, Nandus and its hypertrophy under the influence of the pituitary extracts. The relationship between the pituitary and the ovary has been discussed from various viewpoints and the secretions of the ovarian chromaffin tissue and the interrenal are suggested as the possible mediators in the process of maturation and ovulation in the teleost fishes.     

Evolutionary and Functional Aspects of Pituitary Gonadotropins in the Green Turtle, Chelonia Mydas

Information on the pituitary gonadotropins in Chelonia mydasrepresents some of the most complete data available for anyreptile and thus provides an important basis for evaluatingevolutionary processes in tetrapod endocrine physiology. Thetwo gonadotropins isolated from Chelonia pituitary glands showclear chemical and immunological homologies to mammalian follicle-stimulatinghormone (FSH) and luteinizing hormone (LH). However, receptorstudies and biological tests indicate that the functions ofthese hormones may be different in turtles and mammals. In particular,Chelonia LH shows an unusual ability to interact with FSH receptorsites and to stimulate physiological functions normally attributedto FSH. Results with Chelonia LH demonstrate that errors mayarise from using mammalian hormones to investigate reproductionin turtles. Measurements of endogenous gonadotropin levels inthe plasma of breeding and nesting Chelonia provide a differentperspective of the potential roles of the FSH and LH from thatobtained in physiological tests. In particular, FSH and LH secretionare markedly dissociated during the nesting cycle; FSH has onlya transient peak during oviposition, whereas LH, along withprogesterone, displays a pronounced "ovulatory" surge in theday following nesting. Preliminary studies with synthetic gonadotropicreleasing factors in Chelonia suggest that these may be usefulin inducing reproductive changes.     

Carp maturational-ovulatory gonadotropin but not carp vitellogenic gonadotropin or salmon maturational-ovulatory gonadotropin stimulates testosterone production by rat Leydig cells in vitro

Carp (Cyprinus carpio) maturational-ovulatory gonadotropin, prepared from the fraction of pituitary extract adsorbed on Con A-Sepharose (Con A II) and subsequently adsorbed on CM-cellulose (Whatman CM-52), stimulated testosterone production by isolated rat Leydig cells. The fraction of carp pituitary extract unadsorbed on the immobilized lectin (Con A I) with a mol. wt of 30,000, which had previously been shown to contain vitellogenic gonadotropin, was devoid of steroidogenic activity. Salmon (Oncorhynchus keta) pituitary Con A I and Con A II fractions containing vitellogenic and maturational-ovulatory gonadotropin respectively did not enhance steroidogenesis in the same assay system. The results indicated that carp maturational-ovulatory gonadotropin resembled mammalian luteinizing hormone (LH) in its chromatographic behavior on Con A-Sepharose and CM-cellulose and also in its steroidogenic activity in rat Leydig cells. However, not all teleost maturational-ovulatory gonadotropins are LH-like: the salmon hormone is a notable exception. The data further supports the distinctiveness of carp vitellogenic gonadotropin and maturational-ovulatory gonadotropin.     

Primary culture of normal pituitary cells of carp (cyprinus carpio) for the study of gonadotropin release

Summary A method for preparing enzymaticlaly dispersed pituitary cell cultures of carp (Cyprinus carpio) is described. The cultures have been used to assay a synthetic analog of gonadotropin releasing hormone (GnRH) and to determine the specificity of steroids able to affect gonadotropin (GtH) release in vitro. Time course secretion studies indicated that by 48 h incubation, in the presence of 500 pM GnRH, cumulative secretion of gonadotropin (719 ng±90/2.5×10⁵ cells) had exceeded that of controls (446 ng±106/2.5×10⁵ cells). Estradiol-17β, progesterone, testosterone, and 11-ketotestosterone showed different inhibitory effects on pituitary basal GtH release. Based on the results, it was concluded that carp pituitary cell cultures can be applied to investigations of several aspects of the hypothalamo-hypophysial-gonadal system. This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.     

Molecular analysis and bioactivity of luteinizing hormone from Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>, produced in silkworm pupae

Luteinizing hormone (LH), a gonadotropin hormone (GTH) of the pituitary glycoprotein family, is important in oocyte maturation, ovulation, and spermiation. In this study, we generated a Japanese eel LH (JeLH) with and without the equine chorionic gonadotropin (eCG) carboxyl-terminal peptides under the control of the polyhedron in the silkworm pupae BES to determine their expression levels, glycosylation profile, and in vitro bioactivity. The target proteins were highly expressed in the pupae hemolymph. Recombinant JeLH·eCG and JeLH were N- or O-glycosylated, as shown by periodic-acid Schiff staining, deglycosidase enzyme treatment, and lectin blot analyses, and showed no significant difference in the in vitro bioactivity. Both single-chain hormones and salmon pituitary extract induced maturation of Japanese eel oocytes in vitro. Recombinant LHs produced in silkworm pupae might be suitable candidates for in vivo experiments, because they can be produced in sufficient amount and can undergo N-glycosylation.     

Design and synthesis of potent tyr(OMe)5-gonadotropin-releasing hormone (GnRH) analogues with modifications at positions 6, 9 and 10

We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilic D-amino acids such as D-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.     

Comparative Messenger RNA Expression of FSHβ, LHβ, FSHR,LHR, and ERβ in High and Low Prolific Goat Breeds

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth and maturation, but no clear differences between breeds with different ovulation rates have been found. Therefore, this study investigated mRNA expression of FSHβ, LHβ, FSH receptor (FSHR), LH receptor (LHR), and estrogen receptor-β (ERβ) genes in prolific Lezhi black (LB) goats and nonprolific Tibetan (TB) goats by real-time PCR. Follicles and pituitaries were recovered from goats at 12–24 h after onset of estrus. Real-time PCR analysis revealed that the expression levels of FSHβ and LHβ mRNA were significantly higher (p < 0.01) in pituitary of LB than in TB does, but the expression levels of FSHR and LHR mRNA in follicle of TB were greater (p < 0.05). Expression level of follicular ERβ was not different between the two breeds. Data provide evidence that the greater ovulation rate in the LB goat as compared to the TB breed is associated with a greater gonadotropin expression during follicular phase.     

Effects of Ascorbate on the Oxidation of Derivatives of Hydroxycinnamic Acid and the Mechanism of Oxidation of Sinapic Acid by Cell Wall-Bound Peroxidases

A cell wall fraction isolated from epicotyls of Vigna angularis,which contained both ionically and covalently bound peroxidases,rapidly oxidized p-coumaric, caffeic and ferulic acids and slowlyoxidized sinapic acid. The oxidation of sinapic acid was greatlyenhanced in the presence of p-coumaric, caffeic or ferulic acid.Ascorbate (20 µM) inhibited the oxidation of ferulic acidby about 70% and completely inhibited the oxidation of p-coumaricand ferulic acids. The cell wall fraction was capable of bindingferulic and sinapic acids but not caffeic acid. p-Coumaric acidbound only slightly to cell walls. The oxidation of p-coumaricand ferulic acids by KCl-washed cell walls was inhibited byabout 60% and 10%, respectively, by 20 µM ascorbate, butthe oxidation of caffeic acid was completely inhibited by ascorbateat less than 20 µM. The oxidation of derivatives of hydroxycinnamicacid by peroxidases released from cell walls by washing with1 M KCl was completely inhibited by ascorbate. These resultssuggest that the inhibition by ascorbate depends on the substituentgroup of the phenyl ring of the derivatives of hydroxycinnamicacid when the oxidation reaction is catalyzed by cell wall-boundperoxidases and that the oxidation of sinapic acid is mediatedby phenoxyl radicals of derivatives of hydroxycinnamic acidother than sinapic acid. (Received December 2, 1993; Accepted March 3, 1994)     

The consequences of altered somatotropic system on reproduction

Although the primary control of gonadotropin secretion is by the hypothalamic GnRH and the gonadal function is controlled by the pituitary gonadotropins and prolactin, the emerging evidence suggests a vital role of the somatotropic axis, growth hormone (GH), and insulin-like growth factor-I (IGF-I) in the control of the pituitary and gonadal functions. It has been shown that GH deficiency, GH resistance, and experimental alterations in IGF-I secretion modify folliculogenesis, ovarian maturation, ovulation, and pregnancy, and in the male, GH/IGF-I plays an important role in spermatogenesis and the Leydig cell function. The primary focus of this review is to examine the role of GH/ IGF-I on the onset of puberty, fertility, pituitary, and gonadal endocrine functions. A number of studies have revealed that fertility is affected in GH-deficient dwarf and in IGF-I gene-ablated mice, possibly due to subnormal function of either the pituitary gland or the gonads. In the female GH receptor gene knockout (GHR-KO) mice, there was impairment in follicular development, ovulation rate, sexual maturation, production of and responsiveness to pheromonal signals, and the corpus luteum function. In IGF-I-deficient male GHR-KO mice, puberty is delayed, spermatogenesis is affected, and neuroendocrine-gonadal function is attenuated. Similarly, in some of the human Laron syndrome patients, puberty is delayed due to GH resistance. These data suggest that, in addition to GnRH and gonadotropins, GH/IGF-I influences the pituitary and gonadal functions in animals and humans.     

Purification and some properties of L-tyrosine carboxy-lyase from barley roots

L-Tyrosine carboxy-lyase (E. C. 4. 1. 1. 25) was extracted fromthe roots of barley seedlings and purified approximately 25fold. Optimum pH for the enzyme activity was found to be 7.3.The Km value for L-tyrosine was calulated as 4.5?10^–4M.D-Isomer did not react with the enzyme. L-DOPA, m-tyrosine ando-tyrosine were decarboxylated to some extent. Pyridoxal phosphateactivated the enzyme 4 fold. Caffeic acid and p-coumaric acidare competitive inhibitors. Ki values were 4.5?10^–5M forcaffeic acid and 1.6?10^–4M for p-coumaric acid. L-DOPAand m-tyrosine had an inhibitory effect on the decarboxylationof L-tyrosine. Hydroxylamine, semicarbazide, p-CMB, Fe⁺⁺, Cu⁺⁺,and Hg⁺⁺ inhibited the decarboxylation of tyrosine. Enzyme activitywas also found in extracts from Triticum aestivum, Zea maysand Cytisus scoparius. (Received November 30, 1973; )     

GnRH and GnRH receptors: distribution,function and evolution

Gonadotropin‐releasing hormone (GnRH) was originally identified because of its essential role in regulating reproduction in all vertebrates. Since then, three phylogenetically related GnRH decapeptides have been characterized in vertebrates and invertebrates. Almost all tetrapods investigated have at least two GnRH forms (GnRH1 and GnRH2) in the central nervous system. From distributional and functional studies in vertebrates, GnRH1 in the hypothalamus projects predominantly to the pituitary and regulates reproduction via gonadotropin release. GnRH2, which is located in the midbrain, projects to the whole brain and is thought to be involved in sexual behaviour and food intake. GnRH3, located in the forebrain, has only been found in teleost fish and appears to be involved in sexual behaviour, as well as, in some fish species, gonadotropin release. Multiple GnRH receptors (GnRH‐Rs), G‐protein‐coupled receptors regulate endocrine functions and neural transmissions in vertebrates. Phylogenetic and structural analyses of coding sequences show that all vertebrate GnRH‐Rs cluster into two main receptor types comprised of four subfamilies. This suggests that at least two rounds of GnRH receptor gene duplications may have occurred in different groups within each lineage. Functional studies suggest that two particular subfamilies of GnRH receptors have independently evolved to act as species‐specific endocrine modulators in the pituitary, and these show the greatest variety in regulating neuron networks in the brain. Given the long evolutionary history of the GnRH system, it seems likely that much more remains to be understood about its roles in behaviour and function of vertebrates.