The starting molecule for juvenile hormone (JH) and steroid biosynthesis (Schooley and Baker 1985) is acetyl-coenzyme A (acetyl-CoA) which is converted by the acetoacetyl-CoA-thiolase into acetoacetyl-CoA and by the action of 3-hydroxy-3-methylglutaryl-CoA synthase into (HMG-S) hydroxymethylglutaryl-CoA. From this substance the 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R) generates mevalonate (1; Fig. 6.1). With the mevalonate kinase, the phosphomevalonate kinase and the diphosphomevalonate decarboxylase isopentenyl-pyrophosphate is generated (isopentenyl-PP; 4).

The isopentenyl-isomerase generates finally dimethylallyl-PP (5, Figs. 6.1 and 6.2). From dimethylallyl-PP plus isopentenyl-PP geranyl-PP (6) is derived. Using this geranyl-PP plus isopentenyl-PP the farnesyl synthase builds farnesyl-PP (7; Fig. 6.2 ).

During the JH biosynthesis farnesyl-PP is hydrolyzed to farnesol (8; Fig. 6.2), which is then oxidized to farnesal (9) and finally to farnesoic (aka farnesenic) acid (10). By the JH-methyl transferase the methyl farnesoate (11) is made and by the JH-epoxidase oxidized to the juvenile hormone (13). Alternatively farnesoic acid can be oxidized to the JH III acid and later oxidized to juvenile hormone.

For steroid generation two farnesyl-PP are coupled by the squalene synthase to squalene (14; Fig. 6.4). After epoxidation this squalene is rearranged by the oxido-squalene synthase to lanosterol, which now has the four cyclic rings characteristic for steroids. Lanosterol is oxidized by several oxidation steps to cholesterol, the base of all steroids, or to another compound-like vitamin D₃. Dimethylcholestatrienol (17), made in the ovarian follicle by the lanosterol-14-demethylase (CYP51), is a paracrine stimulator of ovum maturation. From 7-dehydrocholesterol (18) branches off the biosynthesis of vitamin D₃ and related compounds (Fig. 6.4).

The 14-demethylase is the cytochrome P450 enzyme (CYP51) which has been found with identical function in all living phyla: in bacteria, plants, and animals.

JH and steroids are terpenes: monoterpenes are derived from two isopentenyl building blocks, sesquiterpenes¹ such as farnesol of 15 carbon atoms; squalene or lanosterol are triterpenes with 30 carbon atoms.

Juvenile hormones (JH) were discovered almost 50 years ago (Williams 1956; Goodman and Granger 2005; Adams 2003). They are the product of the corpora allata . All ecdysozoan s² have to molt in order to grow. In insects there are, however, two different molts: first the molt between two larval stages (instars) and then the molt to the imago which involves in holometabolic insects pupation. In order to arrive at a molt, the hemolymph ecdysone must increase to high levels. The parameter to decide whether a larval molt or a imaginal molt happens is the JH³: fifth instar larvae of Psacothea hilaris , an east Asian longicorn, may molt—given sufficiently long daylight and warm temperature—to become an imago. However, when treated with JH they have to pass two additional larval molts before becoming an imago (Munyiri and Ishikawa 2004).

The general pathway of JH biosynthesis has already been shown (Fig. 6.3) (for review see: Schooley and Baker 1985). Thus JH III is made (13). Additional JH are JH 0 (26), JH I(25), and JH II(24). These are made only in lepidopterans (butterflies and moths); JH 0 has only been found in eggs of M. sexta . Differences between these three JH and JH III arise from the usage of propionyl-CoA in addition to that of acetyl-CoA. With propionyl-CoA a homomevalonic acid with one extended side chain is formed (21). It depends on a single, double, or triple usage of the derived ethylmethylallyl- and homoisopentenyl pyrophosphate whether JH II, JH I, or JH 0 are made.

As demonstrated in Fig. 6.5 dimethylallyl-PP and isopentenyl-pp (derived from mevalonate) and the derivatives of homomevalonate, ethylmethylallyl-PP (22) and isohexenyl-PP (homoisopentenyl-PP; 23), are species-specifically utilized: animals, which make JH I or JH II, can use both acetyl-CoA and propionyl-CoA. In those synthesizing JH I, the geranyl transferase adds isohexenyl-PP to ethylmethylallyl-PP, and during JH II synthesis, isoprenyl-PP is added to ethylmethylallyl-PP. These steps distinguish JH I and JH II synthesis.

JH diversity is further augmented by side chain hydroxylation, for example, in migratory locust (Fig. 6.6) where any of the three side-chains can be hydroxylated. 12-hydroxy-JHIII (27) tested in mealworms (Tenebrio molitor), was 100-fold more potent than JH III (Darrouzet et al. 1997).

The juvenile hormone of D. melanogaster is a bisepoxide (31; Fig. 6.7). In a first step farnesoic acid it oxidized to 10,11-epoxyfarnesoic acid (12), and in the second step to 6,7;10,11-bis(epoxy)farnesoic acid (30 before being esterified by methyltransferase (Fig. 6.7). In such a pathway any JHIII would not be produced that had not been found in spite of a long search; very new orders of magnitude more sensitive analyses of farnesoates in D. melanogaster showed now that methyl farnesoate and the bisepoxide are present in excess in third instar larvae, whereas in the wandering stage methyl farnoseate in excess of some JHIII and almost no bisepoxide, and in the puparium only methyl farnesoate could be found (Jones et al. 2013). Only in 2009 did Kotaki et al. identify the structure of heteropteran JH as a stereoisomer of the Drosophila JH, and called this bisepoxide juvenile hormone skipped bisepoxide (JHSB₃; 32). The slight difference in the enzyme specificity should help to evaluate the stereospecificity of the epoxidase.

Degradation of JH has two components: hydrolysis by an esterase to the free acid (12) and/or hydrolysis by an epoxide hydrolase to the diol derivative (Fig. 6.8). JH III diol (34)can be further phosphorylated by a kinase (35). Whether diols or diol phosphate are still biologically active has not been analyzed.

In the hemolymph, JH is transported bound to JH binding protein (JHBP).

Insect juvenile hormones have different functions in the larval and the adult animal. In the larval life JH inhibit precocious molt to the adult imago. Up to 15 molts are required in some species before the imago might arise. In the intensively studied butterflies or moths, for example, metamorphosis happens after the fifth instar. It can be postponed by experimental application of JH. For the regulation of JH synthesis stimulating allatotropins, inhibiting allatostatins, and JH-degrading enzymes such as esterases or epoxide hydrolases play their roles (Fig. 6.8).

The interactions of JH and ecdysons are discussed later.

Beyond their role in molting and pupation, JH exhibits several additional functions in the adult animal. In order to arrive at the final molt and at eclosion, the JH level in the hemolymph must be reduced to zero. After eclosion, however, the corpora allata again start JH synthesis and release. Within one day after eclosion JH levels in the hemolymph are again measurable. This JH simulates vitellogenin transcription, which protein is directly involved in female reproductive cycles. JH acts on the fat body. Furthermore, JH facilitates vitellogenin incorporation into competent follicles (Hartfelder 2000).

Crickets (Gryllus rubens ) exhibit two polymorphic types: long-winged, flying (LWF) and short-winged nonflying (SWN) crickets; these differ mainly in JH esterase expression (see Fig. 6.8). Thus it could initially be explained how small differences in JH levels in genetically identical crickets lead to two polymorphic phenotypes. In a particular phase JH titers in SWN crickets were lower than in LWF ones. A time-dependent, diurnal JH concentration in the hemolymph of LWF has been observed with fifteenfold to twentyfold JH increase in the light period compared to dark period whereas in SWN animals no difference due to a dark/light cycle could be found. The picture gets more complex by the finding that prolonged elevated JH levels cause the degradation of flight muscles. It is obvious that there is a narrow time window where LWF have to control their JH concentrations without losing their flight capacity (Zhao and Zera (2004).

In social insects the state of an individual within the colony depends on its hemolymph JH levels. In honeybees (A. mellifera) the different feeding of larvae to become queens and those to become workers induces enhanced JH levels in the queen larvae. These elevated JH levels no longer serve sex organ shaping. The queen suppresses sexual and egg-laying activities in worker bees by release of a pheromone: (E)-9-oxo-2-decenoic acid (Hartfelder 2000). In adult bees, flying activity is stimulated by JH: early in life in queens and drones, later in life for workers to get ready to collect nectar and pollen.

Whether a worker is active in the colony or foraging is determined by the corpora allata and thus most probably JH regulated. With increased corpora allata activity the nutrition forming hypopharynx undergoes a change of function, vitellogenin expression is reduced, and the cerebral mushroom bodies required for sensitive olfaction and orientation are enlarged. Such changes of JH function have only been observed in colony-forming honeybees; in solitary bumblebees, however, or in social wasps JH retains the original gonadotropic functions.

The juvenile hormone receptor has long been evasive. Very recently, the protein methoprene-tolerant has been found to mediate JH actions. Methoprene-tolerant is a bHLH protein that acts as a transcription factor. If methoprene-tolerant is indeed the JH receptor, then this fact would constitute a new type of mechanism of signal transduction: a bHLH transcription factor as receptor inhibiting ecdysone synthesis. Such a novel mechanism would also explain the long search for the receptor (Jindra et al. 2012). The other aspect of this regulation might be the fact that it antagonizes ecdysone-mediated actions and thus is a negative regulator of ecdysone-induced signal cascades.

This enzyme (aka P450 _scc for side chain cleavage or 20,22-lyase) cleaves the cholesterol (19) side-chain between carbon atoms 20 and 22 (Fig. 6.10). The product of this reaction is pregnenolone (37). The earlier name cholesterol monooxygenase indicates that CYP11A1 acts as do all other CYP enzymes by oxidation. The enzymatic complex is assembled by the cytochrome P450 itself, the flavoprotein adrenodoxin reductase , the sulfur–iron-containing adrenodoxin and NADPH as cofactor. The CYP11A1 complex transfers stepwise oxygen molecules onto cholesterol carbon atoms 20 and 22 and takes up the hydrogen atoms, which are stored as water. The necessary electrons for the oxidation stem from NADPH which transfers electrons to the adrenodoxin reductase and in turn reduces adrenodoxin , and then transfers electrons to the cytochrome. From the first oxygen molecule (O₂) one oxygen atom is bound to cholesterol as epoxide, the other atom becomes water. With the second O₂ the epoxide is converted into the diol (plus a water molecule). The third O₂ molecule cleaves the C–C bond and pregnenolone and isocaproic aldehyde are generated, the aldehyde being toxic is removed by the enzyme aldose-ketoreductase 1 B7 (AKR1B7). In very similar ways all CYP enzymes are active. These oxidations are irreversible.

CYP11A1 is an enzyme of the inner mitochondria l membrane (Farkash et al. 1986) as the adrenodoxin reductase . First, StAR has to transfer cholesterol from the cell membrane to the mitochondrium before side-chain cleavage and further steroidogenic conversions can occur.

In GenBank there are sequences from fish, amphibians, reptiles, birds, and mammals. To our surprise two sequences from Trichoplax (an early metazoan precursor) were listed as Cyp11a1. As they are as much related to Cyp24 than to Cyp11a1, the vitamin D₃ hydroxylase (Fig. 6.12) and only have an overall identity of 32 % we do not (yet) consider the putative protein as an active Cyp11a1.

3-Hydroxy-steroid dehydrogenase--isomerase converts pregnenolone (37) to progesterone (38) (Fig. 6.13). During this conversion the hydroxyl group on C-3 is oxidized to a keto group and the double bond between C-5 and C-6 is moved to positions 4 and 5.

Both human 3-HSD isoenzymes are coded for on chromosome 1 and prefer NAD⁺ as cofactor. The type 2 enzyme is preferentially expressed in the adrenal and gonad s, whereas in placenta and other tissues type 1 is found.

The enzyme is not specific for the conversion of pregnenolone to progesterone. In a similar way, 17-hydroxypregnenolone (40) or dehydroepiandrosterone (DHEA (40) are concerted 17-hydroxy progesterone (41) or androstenedione (43) Fig. 6.14).

3-HSD is an enzyme of the smooth endoplasmic reticulum.

In the synthesis steps to cortisol and androgens/estrogens pregnenolone and progesterone are oxidized by CYP17. As the name suggests, the target of this enzyme is the C-atom in position 17 of the sterane/gonane backbone (see Fig. 6.10).

The human CYP17 gene is located on chromosome 10. The enzyme is, like HSD3B, active in the sER . For the conversion of pregnenolone by CYP17 the steroid has to leave the mitochondrion , where it has been generated, and to migrate through the cytosol to the ER.

CYP17 is like CYP11A1 described above, a monooxygenase, and uses electrons and oxygen to oxidize the carbon atom in position 17. The P450 oxidoreductase provides the electrons.

In contrast to other vertebrate-specific CYP enzymes, there are CYP17 in almost any deuterostomes and even in bacteria, making it one of the primordial cytochrom P450 monoxygenases such as CYP51.

17-Hydroxysteroid dehydrogenases belong with the exception of type 5 to the small dehydrogenase/reductase supergene family (SDR). Type 5 is a member of the aldoketo reductase gene family (AKR). Thus far 14 human enzymes with 17-HSD activity have been described. Most of these enzymes possess other catalytic activities apart from 17-dehydrogenase activity. The SDR family of enzymes is of ancient origin and some of the 14 HSD17B genes have homologues in protozoa, plants, and fungi. There are, however, some genes that are not older than the beginning of vertebrate evolution. The SDR enzymes are characterized by the so-called Kallman fold consisting of a bundle of seven parallel sheets, surrounding helices, and binding sites for the NAD/NADP cofactor and steroids.

The intracellular location of these enzymes is diverse. Some of them are cytosolic, some found in microsomes and other vesicles, and others are membrane-bound. It is difficult to see a general rule: whereas 17-HSD type 5 (HSD17B5 ) in the adrenal cortex converts androstenedione (42) into testosterone (44) in the cytosol, the same conversion in testis is performed by 17-HSD type 3 in the endoplasmic reticulum.