Gonadotropin-Releasing Hormone and Its Analogs

Gonadotropin-releasing hormone (GnRH) is a highly conserved hypothalamic decapeptide with the same amino acid sequence in all mammals. After puberty GnRH is released in a pulsatile manner from the hypothalamus, traverses the hypothalamic–hypophyseal portal system, and activates anterior pituitary gonadotroph receptors. The pituitary responds by releasing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a pulsatile pattern.¹ Stimulation of the pituitary by GnRH must be in pulsatile form for repeated release of LH and FSH; after receptor activation, GnRH is rapidly deactivated and cleared. The frequency and amplitude of GnRH pulses vary depending on the phase of the reproductive cycle. The frequency of GnRH pulsatile release in primates during folliculogenesis is every 70 to 90 minutes.² It is this natural pulsatile secretion of GnRH and its short biological half-life that cause difficulties when attempting to use GnRH as a pharmaceutical. Investigations with specialized infusion devices for pulsatile delivery have successfully augmented fertility in a variety of species,^3–5 but the clinical application of such a method lacks practicality for routine use in small animal patients.

GnRH analogs are synthetically prepared substances that differ from GnRH by various amino acid substitutions in the peptide sequence. Analogs with a few amino acid substitutions can act as GnRH agonists because of their increased binding affinity and decreased clearance compared with GnRH. Heavily substituted analogs can cause receptor blockade and have an antagonist function. The result is a suppressive effect on the pituitary–gonadal axis. This axis is easily downregulated such that frequent or high dosing of a GnRH agonist will suppress the release of LH and FSH also.⁶ GnRH agonists and antagonists thus may have the same ultimate physiologic effect.

Oxytocin

A nonapeptide hormone synthesized by neurons in the hypothalamus, oxytocin is transported axonally to the posterior pituitary, where it is stored. This peptide hormone is released from the posterior pituitary into the general circulation after appropriate neural stimulation. Its primary effects are on mammary tissue and the myometrium. The effects of oxytocin on the milk let-down reflex and parturition have been well described.⁷ The ability of oxytocin to induce myometrial contraction is enhanced by prior estrogen sensitization to “prime” the myometrium for maximal response.⁸ The half-life of oxytocin in the blood is short, approximately 1.5 minutes; thus secretion pulse frequency and amplitude are important for its physiologic effects. The release of neurotransmitters may alter the amplitude and frequency of oxytocin release; for example, dopamine enhances burst frequency and amplitude, whereas cholinergic antagonists may be inhibitory.⁹

Luteinizing Hormone and Follicle-Stimulating Hormone

The pituitary gonadotropins LH and FSH are relatively large glycoproteins, each consisting of two covalently bound structural subunits (α and β). Within a species the amino acid sequence of the α-subunits are identical for all anterior pituitary glycoproteins, with high sequence homology existing across species. The β-subunits are specific for individual hormones (i.e., thyroid-stimulating hormone) and provide the functional specificity of each. The overall size of these glycoproteins precludes economic synthetic production of these hormones.⁸

The secretion patterns of these hormones differ depending on the species and the phase of the ovarian cycle. A unique pattern of LH secretion in the bitch has been documented¹⁰ (Figure 23-1). Serum concentration of LH is at basal levels during anestrus. Significant increases in both the amplitude and frequency of LH pulsatile release occur before proestrus. The frequency of LH pulsatile release during anestrus is 3 to 7 hours; before proestrus LH pulse frequency is 60 to 120 minutes.¹¹ This increase in LH release before the onset of proestrus is likely involved with termination of the anestrus phase.¹ The factors that lead to the LH increase at that time are unknown. Serum estrogen concentration at that time also decreases; estrogen production inhibits LH release. What causes the relative decrease in estrogen production at that time is also unknown.

Figure 23-1 Concentrations of luteinizing (LH) hormone in canine serum throughout late anestrus, proestrus, estrus, and early diestrus. Stippled area is the standard error of the mean (bars indicate ranges of proestrus, estrus, and diestrus).

(From Olson PN, Bowen RA, Behrendt MD et al: Concentrations of reproductive hormones in canine serum throughout late anestrus, proestrus, and estrus, Biol Reprod 27:1196, 1982.)

Serum LH concentration returns to basal levels for most of proestrus (i.e., folliculogenesis). The increase in serum estrogen concentration during folliculogenesis contributes to an inhibition of LH release during that phase.¹ When estrogen production decreases, the inhibition of LH release is discontinued. At that time a preovulatory surge in LH release (the preovulatory LH peak) occurs and is thought to trigger ovulation. The duration of the preovulatory LH peak is 1 to 3 days, after which LH secretion returns to a basal state in early diestrus. Additionally, LH is luteotrophic throughout most of the luteal phase in the bitch.

Similar to the pattern of LH secretion, FSH secretion also appears to be inhibited by relatively high concentrations of estrogen during proestrus. Inhibin, a modulary hormone released from the ovary during folliculogenesis, also inhibits FSH release during this phase. When estrogen secretion declines in late proestrus, FSH secretion surges to maximal levels before ovulation, in concert with the preovulatory LH peak^1,10 (Figure 23-2). After this FSH surge, serum FSH concentration remains relatively high during diestrus and pregnancy. Interestingly, the bitch is also unique with regard to the pattern of FSH secretion during anestrus. During anestrus serum FSH concentration can be 50% to 100% of the concentration found at the preovulatory FSH peak and is 5 to 10 times higher than during proestrus.¹ It is unclear why FSH produced during anestrus is unable to stimulate folliculogenesis. It has been postulated that perhaps the FSH measured during anestrus is in a biologically inactive form.¹

Figure 23-2 Concentrations of follicle-stimulating hormone (FSH) in canine serum throughout late anestrus, proestrus, and early diestrus. Stippled area is the standard error of the mean (bars indicate ranges of proestrus, estrus, and diestrus).

(From Olson PN, Bowen RA, Behrendt MD et al: Concentrations of reproductive hormones in canine serum throughout late anestrus, proestrus, and estrus, Biol Reprod 27:1196, 1982.)

The patterns of LH and FSH secretion in the queen have not been clearly determined. It has been documented that after adequate copulation LH release begins within minutes. Queens are induced ovulators; thus this response is expected. Apparent spontaneous ovulation, without copulation or other tactile stimulation, has been reported in the queen.¹² The pattern of LH secretion during the apparent spontaneous ovulation has not been determined, however.

The release of LH and FSH is also pulsatile in response to GnRH in the male. Both glycoprotein hormones are necessary for spermatogenesis. Testicular interstitial cells bind LH and respond by increasing testosterone production. Activation of receptors on Sertoli cells by FSH promotes spermatogenesis and produces inhibin, a hormone that, as in the female, regulates FSH release by the pituitary. After neutering the loss of negative feedback inhibition causes serum FSH concentrations to increase dramatically.¹³ This has also been documented in cases of infertility resulting from primary testicular degeneration.¹⁴ Postcastration elevations in LH serum concentrations also occur, but overlap in measured values between intact and neutered dogs is possible.¹³

Some species also produce placental gonadotropins. A luteotrophic gonadotropin produced by the human placenta (human chorionic gonadotropin [hCG]) has a potent LH-like effect in many species. A gonadotropin produced by fetal trophoblastic cells in horses is referred to as equine chorionic gonadotropin (eCG) or pregnant mare serum gonadotropin (pmSG). In most species eCG has a combination of FSH and LH activity. Additionally, eCG products commonly contain components of equine serum that can be antigenic when used as a pharmaceutical in another species.

Prolactin

A relatively large polypeptide produced by the anterior pituitary, prolactin is luteotrophic in the bitch and queen.^15,16 It is suspected that prolactin is also involved in the maintenance of the anestrus phase in the bitch.¹ Prolactin is under negative control by dopamine such that the administration of a dopamine agonist will inhibit prolactin secretion. Inhibition of prolactin secretion can promote luteolysis during the second half of diestrus or pregnancy in the bitch¹⁵ and queen.¹⁶ Various dopamine agonists have been investigated for inducing abortion and shortening anestrus (i.e., estrus induction). Bromocriptine is a dopaminergic drug that has been used in various protocols. Its use has not gained acceptance, however, because it commonly causes vomiting and diarrhea. The highly potent dopamine agonists cabergoline and metergoline have fewer or no apparent side effects, respectively. The effect of metergoline to decrease prolactin secretion may primarily result from blockade of central serotonin receptors and may function as a dopamine agonist only at higher doses.¹⁷ Metoclopramide, more commonly used as a central serotonin antagonist, may be used to enhance prolactin release in the bitch and queen, and thus enhance milk let-down, through its central dopamine (D₂) agonist effects.

Estrogen

Produced by the ovary during folliculogenesis, estrogens affect target tissues, causing vulvar edema, vaginal mucosal hyperplasia, sanguineous vulvar discharge in the bitch, and sexual attraction in both the bitch and queen. The bitch uniquely exhibits sexual receptivity when estrogen production decreases; serum concentration of estrogen peaks 1 to 2 days before the end of proestrus. A concurrent increase in progesterone is required for complete expression of sexual receptivity. Estrogen acts synergistically with progesterone to stimulate growth of endometrial and mammary glands, and estrogen priming may be important before the natural effect of oxytocin on the myometrium during parturition.⁸ Male production of estrogen occurs in both the Leydig and Sertoli cells, by way of de novo steroidogenesis in the Leydig cell and P450 aromatase conversion of testosterone alone in the Sertoli cell. Considerable interspecies variation occurs with respect to the relative ratios of estrogen produced by each cell. Excessive production of estrogens by Sertoli cell tumors and, less commonly, by other testicular or adrenal tumors results in male feminizing syndrome. Estrogens are involved in receptor sensitivity and feedback influence on the hypothalamic–pituitary–gonadal axis.

The adverse effects of estrogen (i.e., bone marrow aplasia, cystic endometrial hyperplasia/pyometra, infertility) have been documented in dogs after administration of any estrogen preparation.^18,19 The unique sensitivity of dogs to estrogen toxicity may be due to the relatively weak binding affinity of sex-steroid binding proteins for estrogen in the dog. The result is a decrease in the inherent buffering mechanism that would otherwise regulate the amount of free hormone available to penetrate cell membranes. Estrogen toxicity can occur with exogenous or endogenous estrogens (e.g., Sertoli cell tumors, ovarian follicular cysts, and ovarian neoplasia).

Progesterone

As the main progestational hormone in both bitches and queens, progesterone is produced by corpora lutea just before and after ovulation. The bitch continues to produce progesterone during nonpregnant diestrus; serum concentrations of progesterone are indistinguishable in concentration or duration of production from that of pregnancy. The decline of progesterone and increase in prolactin production at the termination of diestrus cause the clinical manifestations of pseudopregnancy in the nonpregnant bitch. This is a demonstration of a normal physiologic occurrence that should not be confused with a pathologic state and in fact suggests normal ovarian function. There is some variation among bitches regarding the maximum amount of progesterone produced in midgestation (or mid-diestrus), with levels reaching 80 to 100 ng/mL in some individuals. The minimum serum progesterone concentration necessary to maintain pregnancy appears to be 2 ng/mL.²⁰ The minimum amount of progesterone required to sustain pregnancy in the queen has not been determined; however, serum progesterone concentrations of less than 1 ng/mL for several days occurred before termination of pregnancy were observed in one study.¹⁶

Placental production of progesterone by the queen during the latter half of pregnancy had been suggested as a requirement to maintain pregnancy. It has been learned, however, that corpora lutea of the queen are necessary for progesterone production throughout pregnancy.²¹ During pseudopregnancy in queens (i.e., nonfertile ovulation), peak luteal activity appears to occur at days 10 to 15 and then declines to basal values by days 35 to 40.²²

In the bitch progesterone is produced before ovulation by follicular luteinization, approximately concurrent with the preovulatory LH peak. Serial evaluations of serum progesterone concentrations during proestrus and estrus can be used to indirectly determine the preovulatory LH peak and thus determine the time of ovulation in the bitch. This methodology, known as ovulation timing, is used to improve breeding management. The bitch typically exhibits sexual receptivity (behavioral estrus) when the serum concentration of estrogen is decreasing and progesterone is increasing.

Synthetic progestational compounds (progestagens) are commercially available. Megestrol acetate has been marketed as a drug to suppress estrus or inhibit ovulation, depending on the time and dose of administration. The method by which progestagens inhibit folliculogenesis and ovulation is not precisely understood. It appears that although megestrol acetate will not decrease the serum concentration of LH that is already at low basal levels, it may be able to prevent the increases in LH that normally occur at the end of anestrus.¹

Testosterone

Produced by the interstitial cells of the testes, testosterone is necessary for gonadal development, spermatogenesis, and libido in the male. The normal function of Sertoli cells to promote spermatogenesis depends on an intratesticular testosterone concentration that greatly exceeds circulatory levels.⁸ Pharmacologic administration of androgens, including testosterone, can induce infertility by negative inhibition of LH and FSH release, which are necessary for spermatogenesis. Testosterone is converted in the prostate to dihydrotestosterone (DHT), which promotes development of this gland and the eventually contributes to the anticipated formation of benign prostatic hyperplasia in mature dogs. DHT is an androgen with greater biological activity than testosterone.

Testosterone and the androgenic steroid mibolerone have been used to suppress ovarian activity and thereby prevent estrus cycles in the bitch. Mibolerone was specifically approved for this use, although the duration of time from discontinuing administration to the occurrence of the next estrous cycle was variable. Silent heats following mibolerone therapy were common. Persistent anestrus (i.e., lack of return to estrous cycles) has been a problem in some Greyhound bitches treated with injectable testosterone to prevent estrus cycles during racing.

Prostaglandin F_2α

Prostaglandins, potent autacoids, have therapeutic indications in small animal theriogenology. Administration of prostaglandin F_2α(PGF_2α) induces a direct luteolytic effect in bitches and queens during pregnancy or diestrus. Induction of luteolysis depends on dose, frequency of drug administration, and stage of diestrus that the drug is administered. After day 30 of diestrus, PGF_2α is reliably luteolytic in both the bitch²⁷ and queen.²² Corpora lutea are relatively resistant to luteolysis by PGF₂-alpha during the first 5 days of diestrus in the bitch.²⁸ There is evidence that PGF_2α will cause either a transient decrease in progesterone production or complete luteolysis when administered during early diestrus after day 6.²⁸ In addition to a direct luteolytic effect, PGF_2α also has a stimulatory action on the myometrium, promoting evacuation of uterine luminal contents. The drug is therefore effective as an abortifacient and for the treatment of open cervix pyometra in the bitch and queen.

Additional effects of PGF₂-alpha administration include panting, nausea, vomiting, diarrhea, hypersalivation, and tachycardia in dogs and vocalization, mydriasis, and possibly vomition and diarrhea in cats. These adverse clinical signs usually stop within 20 to 30 minutes of drug administration. The drug is preferably administered after fasting in the dog and cat to decrease the incidence of vomiting. The adverse effects of PGF_2α are potentially dose related, although there is individual sensitivity, and signs usually abate after repeated dosing. Although extremely rare, cardiovascular collapse after PGF₂-alpha administration is possible.

Most protocols using PGF_2α are based on the formulation of dinoprost tromethamine. This product is not approved for use in dogs or cats in the United States. Informed owner consent is advised, although the use of PGF_2α is established in the veterinary literature. The more potent synthetic PGF_2α analogs (cloprostenol and fluprostenol) are now used clinically in both the dog and the cat; studies regarding the use of these compounds indicate efficacy with fewer side effects, owing to the increased specificity of the compounds for uterine smooth muscle.