Reproductive hormones fall into two broad categories based on their chemical composition - protein and steroid; by definition, these compounds are secreted and gain access to the circulation. Protein hormones are subclassified as neurohormones, gonadotropins, or gonadal proteins. Neurohormones include GnRH, PRF, PIF, oxytocin, and melatonin. Gonadotropic hormones can be of either pituitary (FSH, LH, PRL) or placental (PMSG, hCG) derivation. In addition to steroid hormones, the gonads produce inhibin, relaxin, and MIH. The chemical dispositions, sites of synthesis and action, and most notable biological effects of the reproductive hormones are summarized in Table 2-2. Figure 2-7 portrays a more comprehensive version of the "hierarchy of control" of reproduction than presented earlier (Figure 1-2).

Protein synthesis and secretion. Synthesis of protein hormones (and proteins in general) involves a series of complex steps whereby information contained within the nucleotide makeup of DNA is expressed within a specified sequence of building blocks, the amino acids (Table 2-3); the product is assembled by two processes - transcription and translation.

Transcription involves synthesis of mRNA from a template of DNA. Backbones of the right-handed double helix of DNA are composed of deoxyribose linked by phosphate groups. A purine (adenine [A], guanine [G]) or pyrimidine (cytosine [C], thymine [T]) nucleotide base is attached to each sugar ring. The hydrogen-bonded base pair (A-T, G-C) is the unit of measure of DNA. Transacting nuclear regulatory proteins (transcription factors) dissociate (repressor) or bind (enhancer) to a palindromic sequence of DNA (a two-fold rotational axis of symmetry), activating a consensus cis-element downstream promoter region (TATA, CAT, or GC box). A catabolite activation protein (CAP) binds DNA, prompting the helix to unwind; this allows RNA polymerase to bind to the transcriptional start site and manufacture a complementary single strand of mRNA (ribose replaces deoxyribose; uracil [U] replaces thymine) from antisense DNA.

Genes of eukaryotic (nucleated) cells contain both coding (exons, for EXtra nuclear or EXpressed sequences) and intervening noncoding (introns) regions. Before mRNA is exported from the nucleus, conveying a code for organization of amino acids at the rough endoplasmic reticulum (translation), primary RNA transcripts are cut and spliced into mature mRNAs (ie., introns are removed).

Triplet sequences of bases (codons) provide the genetic information encoded into mRNA that is necessary for polymerization of specific amino acids into a designated sequence. A given amino acid can have more than one codon. There are 61 codons for 20 amino acids in the general code. Ribosomes containing RNA (rRNA) provide structural support for mRNA. Transfer RNAs (tRNA) containing appropriate anticodons transport and attach amino acids to the elongating protein. During translation the ribosome advances step-wise along the strand of mRNA, a single codon (reading frame) at a time, beginning with the initiation codon ATG (which specifies methionine). Synthesis of protein starts at the 5' and terminates at the 3' end of mRNA. Nonsense codons (UAA, UGA, UAG), not corresponding to an amino acid, serve to stop the transcriptional message. A consensus sequence (AATAA) in the 3' untranslated region is the signal for polyadenylation of mRNA. The addition of a poly-A tail is required for mRNA to leave the nucleus (Figure 2-8).

Whereas the DNA content (blueprint) of somatic cells of an individual is identical (excluding random mutations), genes along the helix that can be regulated vary depending upon type of cell; this provides a basis for specialized function among different cells and tissues. In other words, the primary sequences of genes are the same from cell-to-cell - it is the ways they're read that differs. Transcription is suppressed when DNA is bound by protein and rendered inaccessible by a polymerase ("the reader"). The majority of sites along the genome (~ 95%) are masked by histones. Histone (basic) proteins carry a strong positive charge, that attracts DNA. Structures of most nuclear histones are well conserved within and between species. Enzymatic methylation of cytosine bases of DNA (an inherited trait) is also important in the turning on-or-off of specific genes. Inactive genes tend to be highly methylated. The processes of how genes are activated (or suppressed) is called epigenetics.

Protein synthesized along the ribosome is transferred into and through the endoplasmic reticulum and Golgi complex. Translated molecules are often modified in transit through the endoplasmic reticulum and Golgi; this can involve enzymatic modification (eg., cleavage of an active peptide hormone from a larger prohormone), folding (eg., formation of covalent disulfide bonds between residues of cysteine), derivatization (eg., addition of carbohydrate), and(or) subunit assembly.

Protein hormones are packaged into secretory granules within the Golgi apparatus. The cytoskeleton guides (microtubules) and provides a propulsive force (microfilaments) for intracellular transport of granules (Figure 2-9). Secretory granules fuse with the cellular membrane and release their hormonal contents into the extracellular space (exocytosis). Residual hormone is stored in granules and thereby made readily available for secretion upon a subsequent bout of cellular stimulation. Receptors can be processed similarly and incorporated into the cellular membrane (other receptors, enzymes, and structural elements are retained within the cell).

Peptide bonds linking amino acids are susceptible to enzymatic attack. Proteolysis is the major mechanism of inactivation of protein hormones; this can occur within the circulation, liver, kidney, or target tissue. Terminal residues of sialic acid on glycosylated proteins are removed by plasma glycosidases. Hepatic cells recognize the exposed asialo hormone. Protein hormones typically circulate in free form (ie., not bound to transport molecules).

Neurohormones. Vertebrate GnRHs are composed of 10 amino acids. Only one form of decapeptide has been described in eutherian mammals (Figure 2-10). In some species GnRH is found outside the brain (eg., within the gonads [gonadocrinins] and placenta). An extrahypothalamic version of GnRH, with sequence homology to chicken GnRH II, has been reported in marsupials.

Gonadotropin-releasing hormone is cleaved from a prohormone during posttranslational processing. The mature hormone acts on gonadotropes to induce synthesis and secretion of FSH and LH. The first three amino acids of GnRH are involved in activation of a response. Antagonistic analogs of GnRH (bind the receptor, but do not elicit a response) are produced by substituting amino acids in positions 1-3. Amino acids at positions 1 and 10 recognize binding regions on the receptor. Affinity of GnRH for the receptor is amplified by substitution of ethylamide at position 10. Peptide bonds associated with glycine at position 6 are susceptible to enzymatic attack; replacement in this position (eg., with D-Ala) will yield an analog that is resistant to enzymatic hydrolysis (and has a better configuration for interaction with the receptor than native GnRH). Synonyms of GnRH include LHRH, LHRH/FSHRH, gonadoliberin, and luliberin.

There has been interest for some time in the concept that more than one hypothalamic hormone controls FSH and LH. Putative releasing factors specific for FSH, and an inhibitory hormone of LH, have not been purified to homogeneity.

Production of prolactin is controlled by opposing hypothalamic forces. The unequivocal chemical character of prolactin regulating factors has been the subject of debate. Many studies indicate that PIF is dopamine (DA), while others implicate g -aminobutyric acid (GABA). A GnRH-associated peptide (GAP) that is contained within the sequence of the GnRH prohormone also has PIF activity. Thyrotropin-releasing hormone (TRH), vasointestinal peptide (VIP), and 5-hydroxytryptamine (serotonin) cause release of prolactin. The PIF input is of greater physiological significance. A recently discovered pituitary adenyl cyclase-activating polypeptide that belongs to the VIP/secretin/glucagon family stimulates all anterior pituitary cell types.

Oxytocin is composed of nine amino acids; it is assembled on the ribosomes as part of a large precursor that includes its carrier protein, neurophysin. The oxytocin-neurophysin complex is packaged into neurosecretory granules. Neurophysin is cleaved from oxytocin after axonal transport to the neurohypophysis. A disulfide bond links the cysteine residues of oxytocin at positions 1 and 6 (Figure 2-11) - giving the molecule a ring structure that is required for biological activity. Oxytocin stimulates contraction of uterine smooth muscle aiding in expulsion of the fetus during labor and stimulates milk ejection by contracting myoepithelial cells that surround mammary alveoli. Obligatory effects of oxytocin in the male have not been demonstrated. Oxytocin has been isolated within the periphery (gonads, placenta, adrenal gland, uterus, male accessory glands). The plasma half-life of oxytocin is < 5 minutes.

In contrast to the hypothalamic neurohormones described above, melatonin is a product of the pineal gland. The pineal gland is located within the roof of the third ventricle (Figure 2-2). Like the OVLT, the pineal gland is a circumventricular organ, and lies outside the blood-brain barrier (the majority of endothelial cells lining CNS capillaries are sealed by tight junctions, restricting entry of large molecules). Melatonin, an indolamine, is synthesized by enzymatic processing of tryptophan. Tryptophan is converted into serotonin. Serotonin is modified into melatonin by the dual action of two enzymes - N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) (Figure 2-12). Monoamines are inactivated by enzymatic methylation and oxidation. Melatonin is hydroxylated and further conjugated with sulfate. Melatonin participates in the control of seasonal reproduction in some species.

Gonadotropins. Gonadotropins (and TSH) of advanced vertebrates are composed of an a and b subunit. Amino acid sequences of a subunits are well conserved (ie., have evolved from a common ancestral gene). It is the b moiety of gonadotropins that imparts specificity to the molecule (interchange of a subunits with a b yields activity intrinsic to b). Synthesis of mammalian a and b subunits occurs from mRNAs transcribed from genes of different chromosomes. Subunits are glycosylated, compacted by intrachain disulfide bonds, and assembled noncovalently. Synthesis of b subunit is limiting to overall production of hormone. Free b subunit will bind (with reduced affinity) to the gonadotropin receptor; however, glycosylated subunits must be joined to elicit a biological response.

The half-life of the placental gonadotropins within the circulation is longer than for their counterpart pituitary hormones (eg., FSH @ 45 minutes, PMSG = 1-6 days). Additional sialic acid residues shield the placental molecules from metabolic clearance. The placental gonadotropins also have an extended C-terminus (eg., masses of LH and hCG are about 28 and 37 kilodaltons [kDa], respectively).

Human choriogonadotropin (Figure 2-13) binds to luteal LH receptors and stimulates progesterone production - sustaining early pregnancy. Analogous chorionic hormones have been isolated in other primates and the guinea pig. The function of equine choriogonadotropins (horse, donkey, zebra) in pregnancy is indefinite. Horse CG is thought to cause follicular growth and luteinization (ie., formation of accessory or secondary CL) during early gestation (PMSG has both FSH- and LH-like activities).

Prolactin is a single-chain protein that is folded by three disulfide bonds (Figure 2-14); it does not contain residues of carbohydrate. Prolactin is similar in structure to GH and placental lactogens. Prolactin is classified as a gonadotropin in that it stimulates steroidogenic function of the CL in certain species (eg., rodents). Milk synthesis and induction of maternal behavior in farm animals are induced by prolactin. Hyperprolactinemia in humans is associated with infertility, galactorrhea (production of milk outside the normal postpartum period), and gynecomastia (excessive breast development in males); the condition is treated with an ergot alkaloid, cabergoline or bromocryptine (CB-154), dopamine agonists.

Gonadal proteins. Inhibin is a glycoprotein with a molecular mass of approximately 32 kDa composed of two subunits coupled by disulfide bridges. The primary structure of inhibin has been characterized for some species (murine, porcine, bovine, human; Figure 2-15). Inhibin is produced by the testis and ovary. Inhibin of ovarian origin is sometimes called folliculostatin. Two forms of inhibin (A and B) have been isolated. Glycosylated a subunits of each type of inhibin are identical. However, two homologous, but different, unglycosylated inhibin b subunits have been described. Sequences of inhibin subunits are highly conserved between species. Subunits for inhibin are encoded by different mRNAs (probably descendent from one gene). Inhibin is derived from larger precursor molecules. Inhibin is a chalone - a hormone that inhibits rather than stimulates; it specifically suppresses secretion of FSH by acting at the level of the anterior pituitary gland. Diverse patterns of secretion of FSH and LH result from differential control of the gonadotrope imposed by inhibin.

Gonadostatin is a gonadal factor reported to inhibit secretion of both FSH and LH. Several other variants of inhibin have been identified. Dimers formed between inhibin b subunits (activins) exert FSH-releasing properties. Beta subunits of inhibin are structurally similar to transforming growth factor (TGF) b and MIH.

Relaxin is a polypeptide hormone (~ 6000 daltons) produced by the ovaries and placenta; it is made up of two dissimilar subunits (A and B) connected by a pair of disulfide bonds. Three forms of porcine relaxin have been isolated that differ slightly in length of the C terminus of the B chain. Relaxin is synthesized as a preprohormone (Figure 2-16). There is structural homology between relaxin, insulin, and the somatomedins. Structure and potency of relaxin is poorly conserved across species. Relaxin causes dilation of the cervix, relaxation of pelvic ligaments, and coordinates uterine contractions during the birth process.

Mullerian-inhibiting hormone is a disulfide-linked homodimeric glycoprotein of 140 kDa synthesized by the fetal testis. A prepro leader sequence and bioactive C-terminal fragments are cleaved from the mature MIH protein. Regression of the female duct system of the sexually-indifferent male embryo is induced by MIH.

Steroidogenesis. Steroid hormones are derived from cholesterol. Circulatory cholesterol (of dietary origin or synthesized from acetate within the liver) is bound as esters within low density lipoprotein (LDL). Cholesterol-LDL complexes are internalized into the cell by receptor-mediated endocytosis. Receptors oriented within the cellular membrane bind extracellular ligand, move by lateral diffusion, aggregate within discrete zones (such areas are sometimes associated with a coating of clathrin along the inner aspect of the cellular membrane - "coated pits"), and form vesicles that eventually pinch off toward the interior of the cell. Endocytotic vesicles lose their coating and coalesce to form an endosome. The endosome then fuses with primary lysosomes (specialized vesicles of the Golgi complex that contain acid hydrolases) to make a secondary lysosome. Ligand-receptor complexes are dissociated within secondary lysosomes and cholesterol esters are enzymatically cleaved from LDL. Receptors are retained within the vesicle and recycled back to the cellular membrane (Figure 2-17). Fat droplets (Figure 2-18) serve as an intracellular reservoir of cholesterol esters. Only free (hydrolyzed) cholesterol can be used as substrate for biosynthesis of steroid hormones. Cholesterol is transferred to the inner mitochondrial membrane by steroidogenic acute regulatory protein (StAR).

Pregnenolone is derived in mitochondria by enzymatic modification of cholesterol; it is then translocated to the smooth endoplasmic reticulum for further processing into progestogens, androgens, or estrogens. Thus, pregnenolone is a common precursor for gonadal steroidogenesis (Figure 2-19). Primary bioactive secretory products of the CL, testis, and follicle are progesterone (4-pregnen-3, 20-dione), testosterone (17b-hydroxy-4-androsten-3-one), and estradiol (1, 3, 5[10]-estratrien-3, 17b-diol), respectively. The placenta is a major source of steroid hormones during pregnancy.

It has been assumed that steroid hormones are secreted from the cell by passive diffusion (ie., down a concentration gradient). However, there is some evidence that steroids are packaged into secretory granules and released from the cell by exocytosis.

Steroid hormones are only sparingly soluble within plasma. The majority of circulatory steroids (> 90%) are bound to plasma proteins - sex hormone-binding globulin (SHBG; high affinity, low capacity binding) and albumin (low affinity, high capacity binding).

Steroid hormones are metabolized primarily within the liver. The steroid molecule is inactivated by saturation. Conjugation with sulfates or glucuronates makes the steroid hormone water soluble, so it can be excreted (high density lipoprotein apparently targets cholesterol for hepatic metabolism). In some cases circulatory steroid hormones are converted to a more active compound within a target tissue (Figure 2-20).

Gonadal steroid hormones carry out an array of activities - primary effects are on maintenance of reproductive tracts, feedback regulation of secretion of gonadotropins, and behavior. Secondary sex characteristics are also altered by steroids. Female sex steroids interact with other hormones to stimulate growth and development of mammary tissues.

Cortisol is not usually classified as a reproductive hormone; its main actions are in organic metabolism and antiinflammatory/immune responses. Notwithstanding, cortisol originating from the fetal adrenal cortex has been implicated in the triggering mechanism of labor in some farm species. Adrenal 21- and 11b-hydroxylases convert 17a-hydroxyprogesterone to cortisol.