24 Reproduction and Fertility

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From: Endocrinology: Basic and Clinical Principles, Second Edition (S. Melmed and P. M. Conn, eds.) © Humana Press Inc., Totowa, NJ

24 Reproduction and Fertility

Neena B. Schwartz, PhD

C^ONTENTS INTRODUCTION

GONADS AND ACCESSORIES

BRAIN AND PITUITARY

ONTOGENETIC DEVELOPMENT OF REPRODUCTIVE ABILITY

ENVIRONMENT

C^YCLICITY: W^HAT M^{AKES THE} S^YSTEM C^{YCLE IN} F^EMALES? CONTRACEPTION

INFERTILITY

NEW FRONTIERS IN REPRODUCTION

If mating does not occur, this cycle is repeated and mature corpora lutea do not form. Primates run a 28-d menstrual cycle, which includes an active progesterone- secreting luteal phase.

Figure 1 is an illustration summarizing the current understanding of the organs and hormones involved in regulating reproduction in male and female mammals.

Numbers in the figure are cited in the text in parenthesis.

The left side of Fig. 1 represents the components of the system in the female mammal; the right side shows the analogous components in the male.

2. GONADS AND ACCESSORIES

The gonads are characterized by the presence of the germ cells, their accompanying “nurse cells,” and cells that secrete sex-specific steroids into the circulation (see Table 1). Steroid receptors are intracellular, and when the steroid ligand binds the specific receptor in the target organ, within either the cytosol or nucleus, the com- bined entity (transcription factor) binds to specific nuclear DNA and causes transcription of target genes.

Both the ovaries and testes are totally dependent on two peptide hormones secreted by the gonadotrope cells

1. INTRODUCTION

The crucial participation of hormones in reproduction and fertility is the most complicated story in endocrinology, because it involves several organ systems;

gametes as well as hormones; two classes of receptors and intracellular signals; and a myriad of environmental factors such as seasonal signals and, of course, the nearby presence of a conspecific carrier of the opposite gamete type. As complicated as this system is in mammals, being quite different among major classes, it is even more complex when one deals with the vast number of nonmammalian vertebrate species. In a marvel- ous recent review, Rothchild discussed the evolution of placental mammals from other vertebrates. This chapter is limited to two mammals: the rat, which has been the species of choice for elucidating basic science, and the primate, which is obviously of major interest in dealing with clinical issues. The rat runs a 4- or 5-day estrous cycle, from the onset of follicular growth under the influence of follicle-stimulating hormone (FSH), to ovulation following an luteinizing hormone (LH) surge.

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within the anterior pituitary gland: LH (1-1) and FSH (1-2). Specific receptors for these hormones are found within gonadal cell membranes; these receptors are of the seven-transmembrane loop variety, requiring intracellular second messengers to transmit signals to the cell nucleus.

2.1. Testis (1-3)

In the adult male, spermatogenesis is continuous, except in seasonal breeders, with a dividing population of spermatogonia. It takes 40 d in the rat and 70 d in humans for a diploid spermatogonium to become four mature haploid spermatozoa, ready to leave the tubule and move into the epididymis, where they are stored and become mature (1-4). Sertoli cells, the “nurse” cells for future sperm, possess FSH receptors and can aromatize testosterone to estradiol. Testosterone is synthesized and

secreted by the interstitial cells of the testis (1-5), which are found outside the spermatic tubules in close proxim- ity to blood vessels, which empty into the spermatic veins, then carrying blood back to the heart. FSH is necessary for the normal functioning of Sertoli cells; in the absence of FSH, even if LH is present, spermatogenesis does not proceed normally. Spermatogenesis also depends on local high levels of testosterone diffusing from the interstitial cells (1-6). Interstitial cells have LH receptors on their cell membranes and secrete testosterone only when LH is present. Sertoli cells also synthesize and secrete a peptide hormone called inhibin (1-7), which can downregulate FSH synthesis and secretion by pituitary gonadotropes. Testosterone (1-8), acting at the hypothalamus and in the gonadotrope, can suppress LH secretion and, in some cases, increase FSH synthesis and secretion.

Table 1 Gonadal Cell Types

Testis Ovary

Nurse cells Sertoli cells Granulosa cells

Gamete Sperm; renewing Ova; maximum number fixed at birth

Steroid-secreting cells Interstitial cells (hormone: testosterone) Granulosa cells (hormone: estradiol) Sertoli cells (hormone: estradiol) Thecal cells (hormone: testosterone)

Corpus luteum cells (hormone: progesterone) Fig. 1. Illustration summarizing reproductive system in male and female mammals. E = estrogen; P = progesterone; Test = testoster- one; LH = luteinizing hormone; FSH = follicle-stimulating hormone; GnRH = gonadotropin-releasing hormone; GC = granulosa cells; TC = thecal cells; SC = Sertoli cells; IC = interstitial cells; In = inhibin.

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2.2. Ovary (1-9)

Oogenesis stops in mammals before birth, when the oocytes enter the first phase of meiosis. Most oocytes undergo apoptosis (“atresia”) and die between the pre- natal meiotic event and adulthood. The oocytes are encased within the follicles, where they are surrounded by granulosa cells; the outer layer of the follicle con- sists of thecal cells (1-10). Granulosa cells initially express only FSH receptors and the thecal cells express LH receptors. Meiosis resumes in surviving mature oocytes only after the LH preovulatory surge occurs during adult cycles.

Within a given cycle in adults, follicular maturation occurs in a stepwise fashion. Once cycles begin at puberty, a surviving follicle (or follicles, in multiovu- latory species such as the rat) starts to grow, as the granulosa cells divide under the influence of FSH. The thecal cells, under the influence of LH, start to synthesize and secrete testosterone locally (1-10). The testosterone diffuses into the granulose cell layers and is converted into estradiol by the aromatase enzyme in the granulosa cell. As the granulosa cells continue to divide, estradiol secretion into the bloodstream occurs, and estradiol (1-11) begins to act on target tissues and to exert negative feedback on the pituitary and hypothalamus (1-12). Inhibin secretion from granulosa cells also occurs (1-13), and FSH levels fall. The granulosa cells gradually develop LH receptors. Rising levels of estradiol abruptly initiate a rapid rise in gonadotropin- releasing hormone (GnRH) secretion from the hypothalamus (1-14), which causes the preovulatory surges of LH and FSH. After the preovulatory surge of LH occurs, a series of molecular events ensue in the ovary that lead to suppression of estradiol and inhibin secretion, stimulation of progesterone secretion (1-15), and dispersal of the granulosa cells surrounding the ovum.

The ovum then completes the first stage of meiosis, throws off the first polar body, and is extruded from the

follicle (1-16) into the oviduct. If sperm are present fertilization may occur (1-17) and the second polar body is cast off, leaving the fertilized diploid egg; if the uterine environment is favorable, owing to proper action of estradiol and progesterone, implantation of the growing blastocyst occurs in the uterine lining (1-18) after about 4 to 5 d in the oviduct.

3. BRAIN AND PITUITARY

The brain and the anterior pituitary gland are linked, with respect to reproduction, by the secretion of a peptide, GnRH, from the hypothalamus (1-14, 1-21) and the presence of GnRH receptors on the cell membranes of the gonadotrope cells. LH and FSH are dimeric pro- teins, which share a common α-subunit but have different β-subunits, and the entire molecules are recognized by different specific receptors on the cell membranes of the gonads. A pulsatile secretion of GnRH is necessary for continuation of secretion by the gonadotrope cells. Cell lines of GnRH neurons (Gt1 cells) in culture show spontaneous pulses at a frequency of about one per hour. Although GnRH pulses cause secretion of both LH and FSH, differing ratios of the two hormones can be secreted under the influence of alterations in GnRH receptor levels, pulse frequency, and amplitude (Table 2). The GnRH-secreting neurons are found in the arcuate nucleus of the hypothalamus, and GnRH is secreted directly into a portal system that bathes the anterior pituitary cells. LH is more dependent on GnRH than FSH is: increases in GnRH amplitude or frequency enhance LH secretion more than FSH, and GnRH antagonists lower LH more than FSH.

The greater the number of GnRH receptors on gonadotropes, the more LH secretion is favored over FSH.

GnRH receptors (two kinds) are of the seven transmembrane domains and are found in the membranes of the gonadotropes. Most gonadotrope cells synthesize and contain both LH and FSH, although the ratio of the Table 2

Factors Altering Relative LH and FSH Secretion Increase FSH/LH Increase LH/FSH Low-frequency GnRH High-frequency GnRH

Low number of GnRH receptors High number of GnRH receptors

GnRH antagonists Removal of ovaries

Low inhibin Removal of testes

High activin Low follistatin Increased progesterone Increased glucocorticoids Increased testosterone

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hormones within the cells and their distribution across the pituitary varies during the cycle, with maximal levels found just before the LH surge. The second-messen- ger system transducing the GnRH signal to the gonadotrope is highly complex; it involves the mitogen- activated protein kinase pathway and calcium mobiliza- tion. Targets of GnRH activation of its receptor are the genes for the α- and β-subunits of the gonadotropins and the GnRH receptor gene itself.

Progesterone (1-15), testosterone (1-5), and glucocorticoids enhance FSH synthesis when applied directly to pituitaries in culture or in vivo (Table 2). There are also three peptides that are of crucial importance in regulating FSH synthesis and secretion by the pituitary (Table 2). Inhibin is a heterodimer related to the trans- forming growth factor-β (TGF-β) family and is secreted by the ovarian follicles or Sertoli cells, specifically inhibiting FSH synthesis and secretion directly (1-7, 1- 13) in the gonadotrope. Activin is a dimer of the inhibin α-subunit. Activin stimulates FSH synthesis and secretion; activin is made locally in the pituitary gland and is probably as important as GnRH, if not more important, in stimulation of FSH secretion (1-19). The third peptide is follistatin (1-20), which is not homologous to the TGF-β family. It is made in both the ovary and the pituitary gland and acts as an inhibitory binding protein for activin. Follistatin blocks the stimulation of FSH synthesis and secretion from activin, and also from progesterone, testosterone, and glucocorticoids. This suggests that the steroids act on FSH production via stimulation of activin.

Hormonal feedback from gonadal steroids acts at both hypothalamic and pituitary levels (1-8, 1-12) and is primarily negative in nature because removal of the gonads increases GnRH and LH and FSH secretion. The stimulation by estrogen of the preovulatory surges in female mammals is usually labeled “positive” feedback, although if ovulation occurs, ovarian secretion is low- ered as the follicle switches to a corpus luteum, thereby dropping estrogen secretion and favoring progesterone secretion.

GnRH secretion (1-14, 1-21) is regulated by many stimulatory and inhibitory neuromodulators released from interneuron synapses acting at the GnRH neurons. Excitatory inputs include norepinephrine and glutamate (L-glutamic acid), which is the major stimulatory agonist in the hypothalamus. Neuropeptide Y stimulates GnRH release, as well as the action of GnRH in releasing LH from the pituitary, in animals previ- ously exposed to estrogen. γ-Amino- butyric acid is probably the major inhibitory input to the GnRH neurons. Opioids are also inhibitors of GnRH release.

Because GnRH neurons in situ do not contain steroid

receptors, steroids probably act on GnRH release frequency and amplitude by acting on interneurons.

4. ONTOGENETIC DEVELOPMENT OF REPRODUCTIVE ABILITY

In the early embryo, a set of primordial germ cells formed in the placental membranes migrates to the uro- genital ridge, inducing gonad formation with a somatic contribution from the local epithelium. The Y chromo- some contains a gene, sry (sex-determining region of the Y chromosome), which codes for a transcription factor that induces formation of Sertoli cells. In the absence of the sry gene, the somatic supporting cell precursors become follicle cells and proceed toward ovarian development. A number of other genes must also be expressed in order for the formation of normal testis and ovary to take place. Müllerian-inhibiting substance (MIS) is secreted by the testis early and diffuses to the locally forming duct systems; MIS kills the cells of the Müllerian duct, which would have formed into oviduct/uterus. In female embryos, this duct system survives but the Wolf- fian duct system, which is destined to develop into the male accessory ducts, does not survive because it depends on testosterone secretion. Testosterone secretion from the developing testis induces masculinization of the external genitalia. These incipient genitalia cells must convert the testosterone into dihydrotestosterone by means of the enzyme α-5-reductase for this mascu- linizing of the genitalia to take place. For normal female ovarian, duct, and genitalia differentiation to take place, both X chromosomes must be present, and a number of ovarian genes must be expressed.

Normal anterior pituitary gland and hypothalamic differentiation is also necessary for reproduction to develop normally in both male and female genotypes. The cells that secrete GnRH in the mature individual actually are derived embryologically from cells in the olfactory region and migrate during brain development to the hypothalamus. Two “orphan” nuclear receptors (SF1 and DAX1) that appear throughout the hypothalamus, gonadotropes, gonads, and adrenals are heavily involved with normal gonadal differentiation and function in both sexes.

Newborn animals are sexually immature. Puberty occurs when the central drive for GnRH secretion kicks in, and the threshold for negative feedback of gonadal hormones increases. Premature puberty in human males is usually associated with central nervous system mal- function. Reproductive menopause occurs in females when the supply of oocytes remaining in the ovaries becomes inadequate to secrete sufficient estrogen to trigger LH surges. In males, testosterone levels drop with aging, but generally reproduction is attenuated later than in females.

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5. ENVIRONMENT

The connection of the reproductive system to the brain via GnRH provides the conduit whereby the environment provides input to the system. For mammals, there is a value for birth to occur in the spring, when food supplies are most plentiful. Sheep ovulate and mate in the fall, as the ratio of dark to light increases, and with the long gestation period give birth in the spring. Small rodents, by contrast, with a 20-d gestation period, mate and ovulate in the spring, when the light/dark ratio is increasing.

For some species near the equator, because the hours of light and dark remain equal throughout the year, rainfall rather than light may serve as a seasonal signal.

Other species, such as the domestic cat, the rabbit, and the camel, are coitus-induced ovulators. In these species, estradiol stimulates mating behavior, and the cervical stimulus received during coitus triggers a neural reflex that causes a large release of GnRH. This triggers a preovulatory surge of LH that causes ovulation.

6. CYCLICITY: WHAT MAKES THE SYSTEM CYCLE IN FEMALES?

Figure 2 illustrates the changes in the pituitary and ovarian hormones during the nonpregnant rat and primate cycles. The female rat (Fig. 2A) and primate (Fig. 2B) manifest repetitive cycles because the rising levels of estrogen stimulated by background levels of LH and FSH cause the release of a burst of GnRH, which causes the abrupt increase in LH (and FSH) release. These preovulatory surges of LH and of FSH not only cause resumption of meiosis in the most mature follicle(s), but also a cascade of enzymatic changes within the granulosa cells, which terminate estradiol secretion.The resultant corpus luteum begins secreting progesterone. (By contrast, in the male mammal, testosterone and LH levels are maintained at steady levels from day to day, except for the oscillations in both that track GnRH pulses.)

There are two principal operational differences between the rodent and primate cycles. The first is that Fig. 2. Time course of changes in estrogen, progesterone, LH, and FSH during rat and primate nonpregnant cycle: (A) rat estrous cycle. (B) Primate menstrual cycle.

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the rodent cycle is tightly tied to the daily light-dark timing. Not only is the rising estrogen level in the blood a necessary signal for the GnRH release that precedes the LH surge, but there is also a daily circadian signal that occurs between 2:00 PMand 4:00 PMin rats kept in a room lighted from 5:00 AMto 7:00 PM. This neural signal acts in conjunction with the estrogen to closely time the LH/FSH release (Fig. 2A). There is no evi- dence that such a circadian signal operates with estrogen in the primate. The second difference between the rodent and the primate has to do with the luteal phase.

In the rat, mouse, and hamster, there is no spontaneous luteal phase analogous to that in the primate following ovulation. In the primate, the LH surge that triggers ovulation is also adequate to maintain progesterone secretion from the corpus luteum, until placental secretion takes over. In the rat, if pregnancy does not occur, blood levels of both estradiol and progesterone remain low; the resulting absence of steroidal negative feedback (1-12) permits FSH and LH to rise. The prolonged FSH secretion in the rodent cycle (“secondary FSH surge”) (Fig. 2A) occurs because inhibin secretion by the ovary is terminated by the LH surge; this elevated FSH initiates the growth of the next crop of follicles.

However, in the presence of a male, the precedent estrogen secretion followed by the brief proestrous progesterone surge induces sexual receptivity in the female late in the afternoon and mating occurs. The stimulation of the cervix by mating turns on twice daily surges of prolactin in the female, which maintain the progesterone secretion for 12 d or so, permitting implantation.

Pregnancy occurs when a developing embryo implants into the lining of the uterus, prepared by the preceding estrogen and progesterone secretion. In the primate, the corpus luteum formed after the LH surge secretes progesterone spontaneously for about 12 d. For pregnancy to continue, the corpus luteum needs to continue secreting progesterone for about 14 d in the rat and 2 mo in the primate. This steroid is critical for suppressing uterine contractions. Once the embryo is securely implanted in the uterine lining (1-18), the placenta (part maternal, part embryonic) secretes the chorionic gonadotropin necessary to maintain the corpus luteum and eventually also secretes the steroids necessary for main- tenance of the pregnancy and the onset of lactation.

7. CONTRACEPTION

The population of our planet continues to grow ex- ponentially, threatening the environment and outpac- ing food and water supplies. An understanding of the linkages in Fig. 1 is crucial to the design of contracep- tives. The oral contraceptive pill, used by vast numbers of females worldwide, is predominantly progesterone-

like,and suppresses GnRH, FSH, and LH secretion (1- 8), so that ovulation does not occur (1-12). Depopro- vera is a progestin implant that frees females from having to ingest pills on a daily basis. Testosterone implants have been tested in males as a contraceptive;

the high levels of testosterone suppress GnRH, LH, and FSH secretion, thus suppressing spermatogenesis, without depriving the recipient of testosterone necessary for libido and potency (1-8). Condoms block the gametes from meeting and have the advantage of detering the spread of sexually transmitted diseases. In the age of acquired immunodeficiency syndrome and relative sexual freedom, this advantage is extremely important. Tying of the oviducts (1-16) in females or of the vas deferens (1-4) in males, obviously, prevents the gametes from meeting. These simple methods have the advantage of a brief surgery and no drugs with possibly harmful side effects. They are popular in older, stable couples who have completed their families. However, they must be regarded as irreversible, at present. The intrauterine device is a loop that is inserted into the uterus. It alters the uterine luminal environment (1-18) such that implantation cannot take place normally.

Antisera to LH or FSH (1-1, 1-2) have been tested in animals and in some human studies; questions of reversi- bility, side effects, and efficacy are still unsettled.

GnRH antagonists have been tested in men as a contraceptive. Because they reduce LH and FSH (1-21), they also reduce testosterone secretion so potency falls; if they are to be useful they must be accompanied by testosterone.

In females, the “morning after pill,” an estrogen ana- log, diethylstilbestrol, can prevent pregnancy from occurring after unprotected sex, apparently by render- ing the oviductal environment unsuitable for fertilization or survival of the fertilized egg. RU486, an antagonist of the progesterone receptor, can be ingested within a couple of weeks of the onset of pregnancy to cause loosening of the implanted embryo. Following RU486, a prostaglandin-like drug is taken, which initiates uterine contraction, thus expelling the embryo.

8. INFERTILITY

If differentiation of the gonads or the tracts does not occur normally, as in the absence of the sry gene responsible for testicular development, or any of the cascade of genes responsible for steroid synthesis, irreversible infertility results. If chromosomal abnormalities such as XO or XXY occur, the resultant inadequate ovary (XO: Turner syndrome) or inadequate testis (XXY:

Klinefelter syndrome) will result in infertility. Muta- tion of steroid receptors or of peptide receptors in target tissue can cause infertility, by preventing gamete

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maturation, fertilization, or implantation. If the cells that secrete GnRH do not reach the hypothalamus during development, infertility occurs because of

“hypogonadotropic hypogonadism” and is detected when puberty does not occur. Fertility can be induced in such patients by implantation of a pump that injects pulses of GnRH into the bloodstream.

Most frequently, infertility occurs in the presence of normal sexual differentiation, because of failure of the female to ovulate or of the male to have viable sperm. The complexity of the system seen in Fig. 1 sometimes makes it difficult to diagnose the specific site of the problem. In females with normal duct mor- phology, but no spontaneous ovulation, ovulation can be induced by injection of LH or of GnRH. Eggs can be harvested and fertilized in vitro and implanted back into a suitably prepared uterus, or cryopreserved for future use. Artificial insemination by donor sperm can be used by couples when sperm are inadequate in the male. Intracellular sperm injection has also been used when the male partner has inadequate numbers of sperm; a sperm is directly injected into the egg to be fertilized.

9. NEW FRONTIERS IN REPRODUCTION

New technologies have had an impact on reproductive science and practice, just as they have in other areas of biomedical research. However, some of them have been particularly controversial. Molecular biol- ogy techniques have enabled the detection of genetic aberrations that cause some infertility. In vitro fertilization has permitted many couples to bear children, but overproduction of ova with induced ovulation can yield multiple fetuses with resultant health problems. Direct fertilization by means of injection of a single sperm into a harvested ovum has been criticized because of developmental abnormalities detected in some fetuses.

Genetic testing of embryos in utero in order to detect abnormalities has protected some parents from bearing children with extreme abnormalities such as Tay-Sachs disease, but some religious groups oppose the testing.

The sex of a child can now be determined noninvasively

using ultrasound; in some countries such as India, this technique has permitted abortion of “less desirable”

female fetuses, resulting in badly skewed sex pro- portions, which may have social and political implica- tions as the generation of children matures. Stem cell research offers possible tissue harvesting for treatment of disease, such as of insulin-producing cells, but may be employed to create new individuals by reproductive cloning, opening a Pandora’s box of ethical and moral issues. Hormone replacement therapy in postmeno- pausal women has been seen as a boon for women suf- fering extreme hot flashes and has been proven to help prevent osteoporosis. However, there may be risk of breast cancer with the treatment. The subject of reproduction and sex is not a neutral subject sociologically or politically, and knowledge of endocrinology per se is not the last word on how the science can be applied acceptably to these important areas.

24 Reproduction and Fertility