Reproductive Toxicity: Male and Female Reproductive Systems as Targets for Chemical Injury

Environmental Medicine

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Reproductive Toxicity: Male and Female Reproductive Systems as Targets for Chemical Injury Donald R. Mattison, MD, MS,* David R. Plowchalk, PhD,t M. Jane Meadows, PhD);. Amer Z. Al-Juburi, MD,§ Jay Candy, PhD,11 and Antoine Malek, PhD~

Reproductive toxicity has many unique and challenging differences from toxicity to other systems. Whereas other forms of environmental toxicity typically involve development of disease in an exposed individual, because reproduction requires interaction between two individuals, reproductive toxicity will be expressed within a reproductive unit, or couple. 67 This unique, couple-dependent aspect, although obvious, makes reproductive toxicology distinct. For example, it is possible that exposure to a toxic ant by one member of a reproductive couple (e.g., the male) will be manifest by an adverse reproductive outcome in the other member of the couple (e.g., increased frequency of spontaneous abortion). Any attempt to deal with environmental causes of reproductive toxicity must address the couple-specific aspect. There are other unique aspects that reflect the challenges of reproductive toxicology. Unlike renal, cardiac, or pulmonary function, reproductive function occurs intermittently. 5. 6 This means that environmental exposures can interfere with reproduction but go unnoticed during periods when *Professor of Obstetrics and Gynecology, and Interdisciplinary Toxicology. Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, Arkansas tDuke/RJR Postdoctoral Fellow, Toxicology Research Division, RJ Reynolds Co., Wins tonSalem, North Carolina :j:Posldoctoral Fellow, Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, Arkansas §Assistant Professor, Department of Urology, University of Arkansas for Medical Sciences, Little Rock, Arkansas ((Assistant Professor, Division of Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas ~Research Associate, Department of Obstetrics and Gynecology, University of Berne, Berne, Switzerland

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fertility is not desired. This intermittent characteristic can make the identification of a reproductive toxicant in humans more difficult. 7 Another unique characteristic of reproduction, which follows directly from the consideration above, is that complete assessment of the functional integrity of the reproductive system requires that the couple attempt pregnancy. 67 In this summary of reproductive toxicology, we will first explore the definition of successful reproduction. That definition can then be used to characterize what is meant by a reproductive toxicant. The focus of this article will be reproductive toxicity to the sexually mature male and female (Fig. 1); more detailed considerations of reproductive toxicity can be found in several reviews. 2. 4. 24, 34, 40, 87 SUCCESSFUL REPRODUCTION In the sexually mature couple, successful reproduction assumes conception at the appropriate time in their life cycle because of the metabolic, economic, and temporal costs. After conception, the embryo implants within the uterus and develops normally, both structurally and functionally (see Fig. 1). Some abnormalities of fetal development or maternal adaptation to the pregnancy will lead to early pregnancy loss (unrecognized or recognized spontaneous abortion). Successful reproduction also assumes Y1ale Fecundity

Frequency of In lercoursc

I

Conception

f-I--I~_~

Prc-implan ta tion Pregnancy Loss L -_ _ _ _ _ _

~

l--~ _ _l

Female Fecundity

'-----,--------'

Clinical

Pregnancy

Unrecognized Pregnancy

Loss

------1~ _ _1

Term~'~

Clinical Pregnancy Loss

Prc-term Birth "-------"

Figure L Reproductive endpoints vulnerable to environmental influences, Male and female fecundity represent the reproductive competence of the man or woman, After conception, preimpiantation loss may occur. After implantation, pregnancy loss may occur either prior to or after recognition of the pregnancy, Once the pregnancy is established, the possible outcomes include preterm birth, stillbirth, or term birth,

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that the pregnancy progresses to term (38 to 42 weeks), when labor occurs. Correct timing of delivery is essential, because prematurity and postmaturity are associated with increased perinatal morbidity and mortality rates. Finally, successful reproduction requires that the infant adapt to postnatal life and grow and develop normally, including sexually. Successful reproduction is dependent on both individual and couple factors (Table 1). Fecundity is the capacity of a male, female, or couple to produce offspring; fertility is the actual production of offspring. Clearly, the frequency of intercourse should impact on the fertility of a couple. One study by Belsey 7 evaluated the effect of frequency of intercourse on the number of conceptions within 6 months of stopping contraception (Table 2). With intercourse at least four times per week, more than 80 per cent of the couples were able to conceive within 6 months, whereas if intercourse occurred less than once per week, only 16.7 per cent of the couples conceived within 6 months. A similar study by Barrett5 provided data that can be used to define the impact of frequency of intercourse on the cyclespecific fertility, that is, the proportion of couples who conceive within a Table 1. Individual and Couple-Dependent Factors That May Influence Fecundity and Fertility HEI'HODCCTIYE Ei'iDPOIi'iT

FACTOR

Male fecundity

Vasectomy Mumps Feyer Varicocele Diabetes Hypertension Prescription drugs Smoking Alcohol Recreational drugs

Female fecundity

Contraception Tubal Iigation Infection Prescription drugs Smoking Alcohol Recreational drugs Age Ovulatory frequency

Frequency of intercourse

Infections Prescription drugs Alcohol Recreational drugs

Spontaneous abortion

Maternal age Smoking Alcohol Recreational drugs Infection History of spontaneous abortion

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Table 2. Effect of Frequency of Intercourse on Number of Conceptions Within 6 A10nths FREQUENCY OF INTERCOURSE PER WEEK

<1 1 or 2 2 or:1 3 or 4

24

CONCEPTIO/\S WITHI/\ 6 \IO]\;THS (PER CE/\T)

NU\!BER OF CASES

16.7 32.1 46.3

24 109 123 100 72

51.0

83.3

Data from Belsey MA: Infertility: Prevalence, etiology and natural history. In Bracken MB (ed): Perinatal Epidemiology. New York, Oxi()rd Umversity Press, 1984.

given menstrual cycle (Table 3). These data suggest that frequency of intercourse has an impact on fertility, including time to pregnancy and cycle-specific fertility. Therefore, any attempt to define the effect of an occupational or environmental exposure on fertility will clearly have to take frequency of intercourse into account.

EPIDEMIOLOGY OF REPRODUCTIVE FAILURE Given the complex series of events needed for successful reproduction, it is not surprising that reproductive failure should have many possible causes-physiological, infectious, and environmental (see Table 1). In order to appreciate the potential impact of environmental exposures on reproduction, it is necessary to explore the causes and incidence of reproductive failure. Recent data from the National Survey of Family Growth 5l ,52 suggest that 10 to 20 per cent of couples have impaired fertility (Table 4). One way to explore for environmental factors influencing fertility is to look at timedependent trends in infertility. Unfortunately, there are few data addressing this topic (Table 5). Between 1965 and 1982, there has been a three-fold increase in infertility among couples between 15 and 24 years of age. 51 Although there have been smaller increases in infertility over this period among some older groups, these persons do not appear to be as strongly affected. However, given that fertility is highest among younger couples,7 Table 3. Effect of Frequency of Intercourse on Cycle-Specific Fertility FREQUENCY OF INTERCOURSE

Daily Every Every Every Every Every Every

other day third day fourth day fifth day sixth day seventh day

CYCLE· SPECIFIC FERTILITY

0.68 0.43 0.31 0.24 0.20 0.17 0.14

Data from Barrett Je, Marshall}: The risk of conception on different days of the menstrual cycle. Popul Stud 23:455, 1969.

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Table 4. Distribution of Reproductive Status (Per Cent) Among Married Couples AGE OF WIFE FERTILITY STATUS

15-24

25-34

35-44

Surgically sterile Contraceptive N on contraceptive Impaired fertility Fertile

3.5 0.4 10.8 85.4

19.1 6.8 15.5 58.7

27.6 19.4 19.1 33.9

Data from Reproductive Impairments Among Married Couples: United States Survey of Family Growth, Series 23, Number 11. Washington, DC, US Public Health Service, 1987.

this change is of considerable concern. Unfortunately, the cause of this three-fold increase in infertility is not known, although it may suggest an impact of environmental factors on reproduction. In general, the causes of infertility are thought to be roughly one-third male, one-third female, and one-third couple. ,.51. ,52. 67 However, the actual statistical breakdown differs from clinic to clinic (Table 6). Although it is expected that sperm count, sperm motility, sperm morphology, and semen composition all impact on male fecundity,49. 62. 78. 80. 84-86 only sperm count has been clearly demonstrated to have an effect. 45 Female fecundity has been shown in several studies to be influenced by age 4'. 64 (Table 7). Similarly, spontaneous pregnancy loss appears to be influenced by age19 (Table 8). In addition, prior reproductive history has a strong influence on the risk for spontaneous abortion (Table 9). As suggested, reproductive failure is not uncommon. Factors associated with impaired fecundity or fertility or increased risk for spontaneous abortion need to be considered in exploring putative environmental causes of impaired reproduction. Failure to do so may obscure the identification of a reproductive toxicant or falsely identifY a compound as a reproductive toxicant. Both courses have unnecessary costs, both economic and in human health. MECHANISMS OF ACTION OF REPRODUCTIVE TOXICANTS In the simplest sense, reproductive toxicants produce their effects by interrupting the normal flow of matter, energy, or information within or Table 5. Per Cent of Married Women Who Are Infertile (Excluding Surgical Sterilization) YEAR AGE

1965

1976

1982

15-19 20-24 25-29 30-34 35-39 40-44

0.6 3.6 7.2 14.0 18.4 27.7

2.1 6.7 10.8 16.1 22.8 31.1

2.1 10.6 8.7 13.6 24.4 27.2

Data from Fecundity and Infertility in the United States, 1965-1982. National Center for Health Statistics, Advance Data 104. Washington, DC, February 11, 1985.

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Table 6. Causes of Infertility in Couples Evaluated in Infertility Clinics ETIOLOGY

RANGE REPORTED (PER CENT)

Male Azoospermia Oligospermia

20-.50 5-15 15-40

Female Tubal Ovulation Cervix/uterus

25-85 5-85 5-50 5-50

Multiple causes

10-25 0-20

Unexplained

Adapted from Grimes, Richardson: Management of the infertile couple. In Sciarra JJ, Ditts PV (ed): Gynecology and Obstetrics. Chapter 50; and Belsey MA: Infertility: Prevalence, etiology and natural history. Philadelphia, JB Lippincott, 1989. In Bracken MB (ed): Perinatal Epidemiology. New York, Oxford University Press, 1984, pp 255-282.

Table 7. Effect of Age at Marriage on Fertility AGE OF WIFE

FERTILITY RATES

CONCEPTION DELAY

(YEARS)

(PER 1000 WOMEN)

(MONTIIS)

16 17 18 19 20 21 22 23 24 25 26

93 128 121 151 180 209 226 203 276 214 180

11.7 10.4 9.2 8.7 7.2 6.4 6.4 6.0 5.3 6.4 8.9

Data from Belsey MA: Infertility: Prevalence, etiology and natural history. In Bracken MB (ed): Perinatal Epidemiology. New York, Oxford University Press, 1984, pp 255-282.

Table 8. Distribution of Pregnancy Loss (Per Cent) Among Married Couples MATERNAL AGE NO. OF PREGI\AI\CY LOSSES

15-24

25-34

35-44

All

11.6 9.8 1.0 0.8

19.7 14.1 3.7 1.9

31.1 19.9 6.6 4.6

1 2 3+

Data from Mosher WD, Pratt WF: Reproductive impairments among married couples. United States Vital and Health Statistics: National Survey of Family Growth, Series 23, No. 11. Washington, DC, US Public Health Service, 1987.

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Table 9. Recurrence Risk for Spontaneous Ahortion NO. PRIOR EVENTS

Liveborn children

No liveborn children

o

RISK [PER CENT)

12

1 2 3 4

24 26 32 26

2 or more

40-45

Datafrom Simpson: Fetal wastage. In Gabhe JL, Niebyl SG, Simpson JR (eds): Obstetrics: Normal and Problem Pregnancies. New York, Churchill Livingstone, 1986.

between cells or organs in the reproductive system (Fig. 2). This may involve direct interaction with vital cellular components or processes or disruption of endocrine relations essential for reproduction. 42 Direct-Action Reproductive Toxicants Reproductive toxicants can be classified as acting either directly or indirectly on the basis of their mechanism of action. Direct-action reproductive toxicants elicit their effects by virtue of their inherent chemical reactivity or through structural similarity to endogenous compounds such as an agonist or antagonist. Chemical Reactivity. Compounds in the reactivity category are toxic

Direct Acting Reproductive Toxicant

I Metabolism I

Indirect Acting Reproductive Toxicant

Chemical Reactivity Agonist Antagonist Successful Reproduction

Endocrine Disruption

• Matter • Energy • Infonnation Figure 2. Mechanisms of action of reproductive toxicants include direct or indirect pathways that interrnpt the How of matter, energy, or information needed for successful reproduction. Direct-action reproductive toxicants are either chemically reactive or agonistantagonists of endogenous molecules. Indirect-action reproductive toxic ants are either metabolized to direct-action toxicants or disrupt the endocrine system needed for successful reproduction.

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because of their inherent chemical reactivity. They damage macromolecules (DNA, RNA) or organelles (via actions on enzymes and other proteins), disrupting important processes essential for reproductive integrity. Examples are alkylating agents,4. 32. 74 metals, 1.3. 17.29. 3,3. 35. 37. 39. 41. 50. 53. 54. 61. 66. 68, 72. 75.82 organometallics,41. 55 and ionizing radiation. 2 Structural Similarity. Other compounds produce toxic effects through their similarity to biologically important molecules. These compounds thus imitate the action of, or compete for receptors of, an endogenous compound. Moreover, they are capable of triggering inappropriate responses or blocking normal responses in the target cell or organ. These compounds are generally hormone agonists or antagonists that act as competitive inhibitors, although some purine analogues 58 and vitamins are reproductive or developmental toxicants. Probably one of the best examples of this type of compound is the oral contraceptive (estrogen and progestin analogues), which interfere with normal ovarian function by suppressing gonadotropin secretion. 23 By inference, other agents with similar steroid agonist or antagonist properties could alter reproduction by inhibiting gonadotropin secretion; possible examples are D DT and its metabolites,8. 10. 34. 60. 76. 77 Kepone,9. H. 20. 69 cimetidine, and embalming wax. The ability of chemicals to work through this mechanism may also be dependent on temporal changes in the reproductive system. For example, the toxicity of a compound may be limited to a critical period during the ovarian cycle, 15, 18 spermatogenesis, 2. 10, 25 or implantation, If exogenous estrogen or estrogen agonists are administered before a sufficient number offollicle-stimulating hormone (FSH) receptors are formed in antral follicles, the estrogen agonist will suppress pituitary gonadotropin support to the ovary, thereby leading to follicular atresia, 15, 16, 31 Xenobiotics, thought to act as estrogen agonists-antagonists at high doses, are polychlorinated biphenyls (PCBS),14 polybrominated biphenyls (PBBs),63 and organochlorine pesticides.8. ,34, 60 Indirect-Action Reproductive Toxicants Indirectly acting toxicants affect reproductive function either after metabolic activation or by altering normal endocrine homeostasis, Metabolism. In the process of removing a xenobiotic from the body, normal detoxification pathways often generate a number of metabolites, some of which may be more reactive than the parent compounds. The metabolite(s) may then interact by the mechanisms mentioned above for directly acting toxicants (chemical reactivity or structural similarity). Cyclophosphamide is an indirect-action toxicant, requiring metabolic activation by cytochrome P450 mono-oxygenase enzymes before producing chemically reactive metabolites responsible for reproductive toxicity.32 Similarly, polycyclic aromatic hydrocarbons are dependent on microsomal enzymes for bioactivation to metabolites capable of producing reproductive toxicity. 43, 44 Certain halogenated hydrocarbons such as DDT are metabolized to compounds that have estrogenic activity and thus act by the mechanism of structural similarity. 34. 74 Endocrine Homeostasis. Some reproductive toxic ants alter the pulse pattern or circulating levels of hormones important in reproduction. 15. 16,31

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This alteration could entail increasing steroid clearance or elevating or suppressing steroid production. With the recognition that gonadal steroids are essential for feedback loops to the hypothalamus and pituitary for control of normal reproductive function, it became clear that altered circulating levels of steroids could disrupt communication between components of the hypothalamic-pituitary-gonadal axis. IS. 16. :H. 67 Examples of compounds that are capable of inducing metabolic enzyme activities, thus potentially altering endocrine homeostasis, include barbiturates, polycyclic aromatic hydrocarbons, PCBs, PBBs, DDT, and other insecticides that selectively induce enzyme systems.

THE HYPOTHALAMUS AS A TARGET FOR CHEMICAL INJURY The hypothalamus has permissive control of the reproductive system through the pulsatile release of gonadotropin-releasing hormone (GnRH) at a critical frequency and concentration. 105. 16,31.67 The release of GnRH is an intrinsic property of the hypothalamus; however, it is modulated by both stimulatory and inhibitory actions of extrahypothalamic factors (neurotransmitters, progesterone and other steroids). Once released into the hypophyseal portal system, the principal action of GnRB is at the anterior pituitary, where it initiates the synthesis, storage, and secretion of the gonadotropins; FSB, and luteinizing hormone (LlI). Although hypothalamic control is permissive, maintenance of normal gonadotropin levels is dependent on a specific amplitude and frequency of GnRH pulses. Slight deviations seriously alter normal pituitary secretion of gonadotropins. :Jl Disruption of the hypothalamus or the communication pathways to the anterior pituitary results in gonadal atrophy, thus demonstrating the importance of the hypothalamus in maintaining normal reproductive function, albeit through a permissive role. 15, 16. :H The mechanisms by which a chemical might disrupt the reproductive function of the hypothalamus can generally be categorized as any event that could modify the pulsatile release of GnRH. This may involve an alteration in the frequency or amplitude of GnRH pulses. The processes susceptible to chemical injury are those involved in the synthesis and secretion of GnRB, more specifically transcription or translation, packaging or axonal transport, and secretory mechanisms. These processes represent sites where direct-action chemically reactive compounds might interfere with hypothalamic synthesis or release of GnRB. An altered frequency or amplitude of the GnRH pulse could result from disruptions in stimulatory or inhibitory pathways that regulate the release of GnRH. In the process of investigating the regulation of the GnRB pulse generator, it has been shown that catecholamines, dopamine, serotonin, gamma-aminobutyric acid, and endorphins all have some potential for altering the release of GnRH. Therefore, xenobiotics that are agonists or antagonists of these compounds could modify GnRB release, thus interfering with communication with the pituitary.

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THE ANTERIOR PITUITARY AS A TARGET FOR CHEMICAL INJURY Prolactin, FSH, and LH are three protein hormones secreted by the anterior pituitary that are essential for reproduction. 15, 16, 31, 67 They play a critical role in maintaining the ovarian cycle, governing follicle recruitment and maturation, steroidogenesis, completion of ova maturation, ovulation, and luteinization. They are also important in maintaining normal testicular function. The precise, finely tuned control of the reproductive system is accomplished by the anterior pituitary in response to positive and negative feedback signals from the gonads. The appropriate release of FSH and LH during the ovarian cycle controls normal follicular development, and in their absence, amenorrhea and gonadal atrophy ensue. The gonadotropins play a critical role in initiating changes in ovarian follicle morphology and their steroidal microenvironments through the stimulation of steroid production and induction of receptor populations. Timely and adequate release of these gonadotropins is also essential for ovulatory events and a functional luteal phase. Because gonadotropins are essential for ovarian and testicular function, altered synthesis, storage, or secretion of gonadotropins would seriously disrupt reproductive capacity. Interference with gene expression, whether in transcription or translation, post-translational events or packaging, or secretory mechanisms, may modify the level of gonadotropins reaching the gonads. Another mechanism of xenobiotic action is interference with the normal feedback dynamics of ovarian and testicular steroids or other gonadal factors (e.g., protein hormones such as inhibin). Chemicals that act by means of structural similarity or altered endocrine homeostasis might produce their effect by this mechanism. Steroid-receptor agonists and antagonists might initiate an inappropriate release of gonadotropins from the pituitary, thereby disrupting gonadal function. Those chemicals that alter endocrine homeostasis may act by inducing steroid-metabolizing enzymes, thus reducing steroid half-life and subsequently the circulating level of steroids reaching the pituitary,

THE OVARY AS A SITE FOR CHEMICAL INJURY The ovary in primates is responsible for the control of reproduction through its principal products, oocytes and steroid and protein hormones.15, 18 Normal Ovarian Function Folliculogenesis, which involves both intraovarian and extraovarian regulatory mechanisms, is the process by which oocytes and hormones are produced. The ovary itself has three functional subunits: the follicle, the oocyte, and the corpus luteum. During the normal menstrual cycle, these components, under the influence of FSH and LH, function in concert to

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produce a viable ovum for fertilization and a suitable environment for implantation and subsequent gestation. During the preovulatory period of the menstrual cycle, follicle recruitment and development occur under the influence of FSH and LH. The latter stimulates the production of androgens by thecal cells, whereas the former stimulates the aromatization of androgens into estrogens by the granulosa cells and the production of inhibin, a protein hormone. Inhibin acts at the anterior pituitary to decrease the release of FSH. This prevents excess stimulation of follicular development and allows increased development of the dominant follicle-the follicle destined to ovulate. Estrogen production increases, stimulating both the LH surge, resulting in ovulation, and the cellular and secretory changes in the vagina, cervi~, uterus, and oviduct that enhance spermatozoa viability and transport. In the postovulatory phase, the thecal and granulosa cells remaining in the follicular cavity of the ovulated ovum form the corpus luteum and secrete progesterone. This hormone stimulates the uterus to provide a proper environment for implantation of the embryo if fertilization occurs. When the ovum is not fertilized, the corpus luteum regresses, progesterone concentration decreases, and menstruation occurs. Unlike the male gonad, the female gonad has a finite number of germ cells at birth and is therefore uniquely sensitive to reproductive toxicants. Such exposure of the female can lead to decreased fecundity, increased pregnancy wastage, early menopause, or infertility, depending on the component affected, the magnitude of the damage, and the timing of the exposure. 2, 4, 41-43 As the basic reproductive unit of the ovary, the follicle maintains the delicate hormonal environment necessary to support the growth and maturation of an oocyte. As noted, this complex process is known as folliculogenesis and involves both intraovarian and extraovarian regulation, Numerous morphologic and biochemical changes occur as a follicle progresses from a primordial follicle to a Graafian follicle, and each stage of follicular growth exhibits unique patterns of gonadotropin sensitivity, steroid production, and feedback pathways. These characteristics suggest that a number of sites are available for xenobiotic interaction. Also, there are different follicle populations within the ovary, which further confounds the situation by allowing for differential follicle toxicity, This creates a situation in which the patterns of infertility induced by a chemical agent would depend on the follicle type affected, For example, toxicity to primordial follicles would not produce immediate signs of infertility but would ultimately shorten the reproductive lifespan. On the other hand, toxicity to antral or preovulatory follicles would result in an immediate loss of reproductive function. The follicle complex is composed of three basic components: granulosa cells, thecal cells, and the oocyte, Each of these components has characteristics that may make it uniquely susceptible to chemical injury. The following sections describe these individual components and the possible sites of action of reproductive toxicants. 59 Granulosa Cells as Targets for Chemical Injury In their role as a component of the follicle, as well as the supporting cell for oocytes, granulosa cells have several sites of vulnerability to chemical

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injury (Table 10). Gonadotropin receptors are essential for the integration of hormonal signals by granulosa cells. Chemicals that are gonadotropin antagonists, damage gonadotropin receptors, or uncouple the receptor from other molecules essential for hormone action will clearly have an adverse effect on granulosa cell function. For example, a toxicant that is a gonadotropin antagonist and blocks access to the receptor will impair FSHstimulated estrogen synthesis during the follicular phase of the cycle. Several investigators have explored methodology for screening xenobiotics for granulosa cell toxicity by measuring the effects on progesterone production by granulosa cells in culture. 22 Estradiol suppression of progesterone production by granulosa cells has been utilized to verify granulosa cell responsiveness. The pesticide p,p'-DDT and its o,p'-DDT isomer produce suppression of progesterone production apparently with potencies equal to that of estradiol. By contrast, the pesticides malathion, parathion, and dieldrin and the fungicide hexachlorobenzene are without effect. 22 Further detailed analysis of isolated granulosa cell responses to xenobiotics is needed to define the utility of this assay system. The attractiveness of isolated systems such as this is economy and ease of use; however, it is important to remember that granulosa cells represent only one component of the reproductive system. Thecal Cells as Targets for Chemical Injury Thecal cells provide precursors for steroids synthesized by granulosa cells (Table 11). Thecal cells are believed to be recruited from ovarian Table 10. Granulosa Cells as Targets for Chemical Injury SITE OF ACTION

MECHANISM OF ACTION (OUTCOME)

FSH and LH receptors

Decreased receptor population Competition for receptor U ncou pling of receptor to secondary messenger (Decreased estradiol production) (Accumulation of androgens --> atresia) (Inadequate luteinization) (Decreased progesterone production) (Luteal phase defects)

Steroid production

Altered estrogen production: inhibited or depressed aromatase activity (Excessive follicular androgens --> atresia) Inadequate source of androgens (Decreased estrogen --> altered follicle growth) Altered progesterone production: inhibition of enzymes responsible for biosynthesis of progesterone from cholesterol Inadequate luteinization of granulosa cells (Decreased progesterone --> inhibition of FSH) (Decreased progesterone --> luteal phase defect)

Cell proliferation

General cytotoxicity Mitotic inhibitors Reduced production of growth factors (Follicular atresia)

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Table H. Thecal Cells as Targets for Chemical Injury SITE OF ACTION

MECHANIS~l

OF ACTION (OCTCOMEI

LH receptors

Decreased receptor population Competition for receptor Uncoupling of receptor from secondary messenger (Decreased androgen biosynthesis) (Insufficient substrate for granulosa cells) (Altered follicular growth)

Steroid production

Inhibition of enzymes responsible for biosynthesis of androgens from cholesterol (Altered androstenedione and testosterone levels) (Insufficient substrate for granulosa cells)

Cell proliferation

Disrupted migration of stroma to form thecal cell layer General cytotoxicity Mitotic inhibition (Reduced production of growth factors)

stroma cells during follicle formation and growth. Recruitment may involve stromal cellular proliferation as well as migration to regions around the follicle. Xenobiotics that impair cell proliferation, migration, and communication will impact on thecal cell function. Xenobiotics that alter thecal androgen production may also impair follicle function. For example, the androgens metabolized to estrogens by granulosa cells are provided by thecal cells. Alterations in thecal cell androgen production, either increases or decreases, are expected to have a significant effect on follicle function. For example, it is believed that excess production of androgens by thecal cells will lead to follicle atresia. In addition, impaired production of androgens by thecal cells may lead to decreased estrogen production by granulosa cells. Either circumstance will clearly impact on reproductive performance. At present, little is known about thecal cell vulnerability to xeno biotics. Oocytes as Targets for Chemical Injury Although there is a paucity of information defining the vulnerability of ovarian cells to xenobiotics, there are data clearly demonstrating that oocytes can be damaged or destroyed by such agents. Multiple sites for chemical injury are present in the oocyte (Table 12). Alkylating agents destroy oocytes in humans 4 . 32, 74 and experimental animals. 65 Lead produces ovarian toxicity characterized by follicular atresia in rodents and nonhuman primates.-54 , 61, 72 Other metals, including mercuryl7, 28, 35, 39, 41, 50, 68 and cadmium, 29,53,75 also produce ovarian damage that may be mediated through oocyte toxicity. THE TESTIS AS A TARGET FOR CHEMICAL INJURY Normal Testicular Function The human testis is an ovoid mass averaging 32 gm, 4.5 cm in length, with a volume of 25 cc that lies within the scrotum. The testis is surrounded

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Table 12. Oocytes as Targets for Chemical Injury SITE OF ACTION

MECHANIS~I

OF ACTION (OUTCOME)

Oocyte maturation

Disrupted communication between oocyte and granulosa cells of the corona radiata (Loss of proper biochemical signals for maturation) Interference with synthesis and secretion of the zona pellucida proteins (Abnormal sperm receptor content ~ nonviable ovum) General cytotoxicity to cellular processes (Oocyte death)

Meiotic maturation

Damage to oocyte DNA Disrupted communication with granulosa cells Interference with mechanisms that control germinal vesicle breakdown (U ntimely meiotic divisions)

by a connective tissue capsule made up of three tunica layers. The testis receives approximately 9 ml of blood per 100 gm of tissue per minute. The testicular temperature is 2 to 4°C lower than the rectal temperature. The bulk of the testicular tissue consists of seminiferous tubule, and the rest is interstitial consisting of blood vessels, lymph vessels, fibroblastic supporting cells, macrophages, and mast and Leydig cells. The interstitial tissue occupies about 12 per cent of the testicular volume. Testosterone is the principal testicular steroid, and the Leydig cell is its intratesticular source. Testosterone plays an important role in many male reproductive functions (Fig. 3). The seminiferous tubules are long U-shaped tubules, both ends of which usually terminate in the rete testis. It is estimated that the total length of seminiferous tubules in the human is 255 m. 38 The seminiferous tubules contain germinal elements and supporting cells. The supporting cells include the sustentacular cells of the basement membrane and the Sertoli cells. The germinal elements are a slowly dividing primitive stem cell population, the rapidly proliferating spermatogonia, spermatocytes undergoing meiosis, and metamorphosing spermatids. The Sertoli cell is a static non proliferating cell characterized by its irregularly shaped nucleus, prominent nucleolus, filamentous cytoplasmic projection, lack of connections with germ cells, low mitotic index, and unique tight junctional complexes between adjacent Sertoli cell membranes (the principal site of the blood-testis barrier). The functional significance of this barrier remains to be defined. A practical consideration is the possibility of differential xenobiotic access to those cells sequestered behind the barrier, namely the basal compartment of the seminiferous tubule. The Sertoli cells are involved in the production of estradiol from testosterone and the production or concentration of an androgen-binding protein. The germinal epithelium is responsible for spermatogenesis, which takes approximately 65 days. During spermatogenesis, there are at least 13 recognizable germ-cell types. 10, 25 The human testis produces billions of spermatozoa daily throughout several decades of reproductive life. The

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Differentiation of Male Reproductive Organs

Organization of eNS, and Accessory Organs 'vIeta bolic

Testosterone

Gencral Sex Charact.eristics (Body size, hair growth, pigrrl('n la jj on)

Activation

Androgenic

Sexual Dehavior, Accessory Sex Organs, Spernlatogenesis

Figure 3. Actions of testosterone in the male.

testes of hypophysectomized men are characterized by Leydig cell atrophy, peritubular hyalinization, and germinal cell depletion that ranges from tubules containing only spermatogonia to those with scattered spermatocytes. Follicle-stimulating hormone and interstitial cell-stimulating hormone (ICSH or LH) are the principal pituitary hormones responsible for regulation of spermatogenesis. Together, FSH and LH produce a greater response in the initiation of spermatogenesis in pubertal males and the reinitialization of spermatogenesis in hypophysectomized animals than either gonadotropin added alone. Luteinizing hormone probably exerts its effect on spermatogenesis by increasing the production of testosterone via an increase in the rate of conversion of cholesterol to pregnenolone, thus providing more pregnenolone for conversion to testosterone. Testosterone is further metabolized by the testis to estrogens or dihydrotestosterone. This increased production of testosterone is apparently required for meiosis. In contrast, the mechanism by which FSH affects spermatogenesis is largely unknown. Even though chemicals toxic to the male reproductive tract may act on a number of different cellular targets, the ultimate manifestation of the effects will likely be reduced sperm production and decreased fertility. 78, 79, 84-86 Because semen can easily be obtained noninvasively, analysis of human sperm has been the predominant tool for assessing possible chemical influences on male reproductive function. Effects of Toxicants on Testicular Function Some therapeutic drugs affect testicular function and diminish fertility, mainly by producing oligospermia. This could happen by an indirect effect through the hypothalamic-pituitary-gonadal axis or by a direct insult to

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the testis as a target organ. Many of these effects are reversible with cessation of the drug. Diethylstilbestrol or any other kind of medicine with an estrogen-like effect impairs testicular function, and men treated with diethylstilbestrol have atrophic seminiferous tubules. Many steroids produce an effect on fertility either by delaying spermatogenesis or by stopping it completely. Nitrofurantoin and sulfasalazine lead to oligospermia. Although there was no proof of an effect on spermatogenesis with the use of cimetidine, there was an increase in the number of benign Leydig-cell tumors in laboratory animals given the drug. Usage of drugs with antiandrogen activity is not recommended in men with a fertility problem. The largest group suffering from infertility are the recipients of modern cancer chemotherapy. Many of these agents, such as cyclophosphamide, either severely impair spermatogenesis or arrest it completely. The dim chance of recovery comes only many years after the treatment. 2 Historically, investigations of chemical effects on male reproductive function have been directed principally toward therapeutic agents. However, in the mid-1970s, reports began to emerge linking occupational exposure to reduced male reproductive capacity (Table 13). Workers involved in the manufacture of the nematocide 1,2-dibromochloropropane (DBCP) experienced infertility, the frequency being dependent on the Table 13. Human Sperm Studies of Occupational Exposures SPERM INDlCATOR*

Count Motility Morphology F Bodies

Detrimental Effects Carbon disulfide Dibromochloropropane (DBCP) Lead Inconclusive Boron Cadmium Carbaryl Ethylene dibromide Kepone Methylmercury Toluene diamine and dinitrotoluene No Effects Anesthetic gases Epichlorohydrin Ethylene glycol monomethyl ether Formaldehyde Glycerine production Para-tertiary butyl benzoic acid Polybrominated biphenyls Waste water treatment

+ + +

+ +? +

+ +? +

+? +? +? +? +? +?

+? +?

+?

+?

+

REFERENCES

36.48 81 37 33 66 80 86 70 9 55 21

84 49 12 73 71 81 63 62

* + = detrimental effect observed; +? = detrimental effect with unknown or marginal statistical significance or not clearly related to the exposure; - = no detrimental effect observed.

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length of employment and extent of exposure (i. e., time and dose dependence). The chemical alters spermatogenesis, resulting in decreased sperm counts, increased serum FSH and LH, and reduced fertility. 78. 79 Animal experiments suggest that DBep acts on interstitial cells of the testes, disrupting either the action or the production of androgen. 56.57 Kepone (chlordecone) is an insecticide that has been associated with male infertility following occupational exposures. 9 , 11,69 Extended exposure to Kepone produces testicular effects manifested as low sperm counts and reduced sperm motility. 8 These effects are thought to be secondary to estrogenic-like activity of Kepone on the testes. 16 It should be noted that extended Kepone exposure induces delayed neuropathy in peripheral long axons of the body, and for this reason, this compound is no longer marketed. Male radiotherapy patients and volunteers receiving graded radiation doses to the testes during controlled experiments display temporary aspermia or oligospermia, with the time required for recovery being dependent on the dose and duration of exposure. 2 The reduced sperm count will take several weeks to be manifested because the developing type A spermatogonia are the sensitive cell type. The later spermatogenic stages are more radioresistant and therefore maintain the sperm count in the normal range until the damaged developmental stages would have reached maturity. Recovery from oligospermia may take several months or even years. Although studies present conflicting results, chronic alcohol abuse has been associated with abnormalities of spermatogenesis and presumed subfecundity. Males exposed to diethylstilbestrol in utero commonly display abnormal spermatozoa and reduced fertility, Although this effect remains in some dispute, potential mechanisms include alteration in the development of the male reproductive system leading to either structural or functional abnormalities of the accessory organs, especially the epididymis and vas deferens. Marijuana has been reported to reduce serum testosterone, LH, and FSH concentrations in rodents. However, the results from human experiments are equivocal, and later reports have not been able to confirm earlier studies suggesting that chronic smoking of marijuana alters steroid hormone levels. 26,46 Several controlled studies in humans have documented decreased sperm counts and reduced sperm motility after high-dose marijuana exposure. 26, 46 Methyl chloride treatment of rodents results in altered sperm characteristics. This effect has been attributed to chronic inflammation of the epididymis. 27 No reports have been found of reproductive toxicity to men by methyl chloride. SUMMARY On the basis of current knowledge of reproductive biology and toxicology, it is apparent that chemicals affecting reproduction may elicit their efiects at a number of sites in both the male and the female reproductive system. This multiplicity of targets is attributable to the dynamic nature of

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the reproductive system, in which the hypothalamic-pituitary-gonadal axis is controlled by precise positive and negative feedback mechanisms among its components. Interference by a xenobiotic at any level in either the male or the female reproductive system may ultimately impair hypothalamic or pituitary function. Normal gonadal processes such as spermatogenesis or oogenesis, ejaculation or ovulation, hormone production by Leydig or granulosa cells, and the structure or function of the accessory reproductive structures (e. g., epididymis, fallopian tube) also appear vulnerable to xenobiotics. The reproductive system is a complex one that requires local and circulating hormones for control. This brief review illustrates a system for characterizing the mechanism of action of reproductive toxicants, as well as for defining the sites available for disruption of reproduction. Unfortunately, at present, data addressing the actual vulnerability of reproduction are sorely lacking. However, when experiments have been conducted and combined with epidemiologic data or clinical observation, it has been possible to demonstrate impairment of reproductive processes by xenobiotics. The role of environmental exposure to xenobiotics in the increase in infertility that has been observed remains to be defined.

REFERENCES 1. Arenholz MS, Meyer CR: Health hazard evaluation determination report, Olin Chemical Company, Bradenburg, Kentucky. Publication HE 70-113-728. Washington, DC, National Institute of Occupational Safety and Health, 1980 2. Ash P: The influence of radiation on fertility in man. Br J Radiol 53:271, 1980 3. Baird DD, Wilcox AJ: Cigarette smoking associated with conception delay. JAMA 253:2979, 1985 4. Barber HRK: Tbe effect of cancer and its therapy upon fertility. Int J Fertil 26:250, 1981 5. Barrett JC: Fecundability and coital frequency. Popul Stud 25:309, 1971 6. Barrett JC, Marshall J: The risk of conception on different days of the menstrual cycle. Popul Stud 23:455, 1969 7. Belsey MA: Infertility: Prevalence, etiology, and natural history. In Bracken MB (ed): Perinatal Epidemiology. New York, Oxford University Press, 1984, pp 255-282 8. Bitman J, Cecil HC: Estrogenic activity of DDT analogs and polychlorinated biphenyls. J Agr Food Chem 18:1108, 1970 9. Cannon SB, Veazey JM Jr, Jackson RS, et al: Epidemic Kepone poisoning in chemical workers. Am J Epidemiol 107:529, 1978 10. Clermont Y: The cycle of the seminiferous epithelium in man. Am J Anat 112:35, 1963 11. Cohn WJ, Boylan JJ, Blanke RV, et al: Treatment of chlordecone (Kepone) toxicity with cholestyramine: Results of a controlled clinical trial. N Engl J Med 298:579, 1978 12. Cook RR, Bodner KM, Kolesar RC, et al: A cross-sectional study of ethylene glycol mono methyl ether process employees. Arch Environ Health 37:346, 1982 13. Der R, Fahim Z, Yousef M, et al: Effects of cadmium on growth, sexual development, and metabolism in female rats. Res Comm Chem Pathol Pharmacol 16:485, 1977 14. Derr SK, Decker J: Alterations of androgenicity in rats exposed to PCB's (Aroclor. 1254). Bull Environ Contam Toxicol 21:43, 1979 15. diZerega G, Hodgen GD: Folliculogenesis in the primate ovarian cycle. Endocrinol Rev 2:27, 1981 16. Fink G: Gonadotropin secretion and its control. In Knobil E, Neill JD (eds): The Physiology of Reproduction. New York, Raven Press, 1988, pp 1349-1377 17. Goncharuk GA: [Problems of occupational hygiene in women engaged in mercury production.] (Russ). Gig Tr ProfZaboI21(5):17, 1977

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Ill. Goodman AL, Hodgen GO: The ovarian triad of the primate menstrual cycle, Recent Prog Horm Res 39:1, 19113 19. Currero VR, Rojas OI: Spontaneous abortion and aging of human ova and spermatozoa. l\' Engl J Med 293:573, 1975 20. Guzelian PS: Comparative toxicology of chlordecone (Kepone) in humans and experimental animals. Annu Rev Pharmacol Toxicol 22:89, 1982 21. Hamill PVV, Steinberger E, Levine RJ, et al: The epidemiologic assessment of male reproductive hazard from occupational exposure to TDA and DNT. J Occup Med 24:98.5, 1982 22. Haney AF, Hughes SF, Hughes CL Jr: Screening of potential reproductive toxicants by use of porcine granulosa cell cultures. Toxicology 30:227, 1984 23. Harrington JM, Stein GF, Hivera RO, et al: Occupational hazards of formulating contraceptives: A survey of plant employees. Arch Environ Health 33:12, 1978 24. Heinrichs WL, Juchau MR: Extrahepatic drug metabolism: The gonad. In Gram TE (ed): Extrahepatic Metabolism of Drugs and Other Foreign Compounds. New York, SP Medical and Scientific Books, 1980, pp 313-332 25. Helier CG, Clermont Y: Kinetics of the germinal epithelium in man, Hecent Progr Horm Hes 20:545, 1964 26. Hembree WC Ill, l\'ahas GG, Zeidenbert P, et al: Changes in human spermatozoa associated with high dose marijuana smoking. Adv Biosc 22 and 23, 1979 27. Hurt ME, Working PK: Evaluation of spermatogenesis and sperm quality in the rat following acute inhalation exposure to methyl bromide, Fund Appl Toxicol 10:490, 1988 28. Jagiello G, Lin JS: An assessment of the effects of mercury on the meiosis of mouse ova. ~lutat Res 17:93, 1973 29. Kar AB, Das HP, Karkun IN: Ovarian changes in prepubertal rats after treatment with cadmium chloride. Acta Bioi Med Germ 3:272, 1959 30. Kimbrough RD: The toxicity of polychlorinated polycyclic compounds and related chemicals. Crit Rev Toxicol 2:445, 1974 31. Knobil E, Hotchkiss J: The menstrual cycle and its neuroendocrine control. In Knobil E, Neill JD (eds): The Physiology of Reproduction, vol 2. New York, Raven Press, 1981l, pp 1971-1994 32. Koyama H, Wada T, l\'ishizawa Y, et al: Cyclophosphamide-induced ovarian failure and its therapeutic significance in patients with breast cancer. Cancer 39:1403, 1977 33. Krasovskii GN, Varshavskaya SP, Botisov AI: Toxic and gonadotropic effects of cadmium and boron relative to standards for these substances in drinking water. Environ Health Perspect 13:69, 1976 34. Kupfer 0: Effects of pesticides and related compounds on steroid metabolism and function. Crit Rev Toxicol 4:83, 1975 35. Lamperti AA, Printz RH: Effects of mercuric chloride on the reproductive cycle of the female hamster. BioI Reprod 8:378, 1973 36. Lancranjan I: Alterations of spermatic liqUid in patients chronically poisoned by carbon disulfide. Med Lav 63:29, 1972 37. Lancranjan I, Popescue HI, Gavanescu 0, et al: Reproductive ability of workmen occupationally exposed to lead. Arch Environ Health 30:396, 1975 38. Lennox B, Ahmad KN: The total length of tubules in the human testis. J Anat 107:191, 1970 39. Marinova G, Chakarova 0, Kaneva I: Study of the reproductive function in women in contact with mercury. Probl Akush Ginekol (Sofia) 1:75, 1973 40. Mattison OR: The effects of smoking on reproduction from gametogenesis to implantation. Environ Res 28:410, 1982 41. Mattison OR: Ovarian toxicity: Effects on sexual maturation, reproduction and menopause. In Clarkson TW, Nordberg GF, Sager PR (eds): Reproductive and Developmental Toxicity of Metals. New York, Plenum Press, 1983, pp 317-342 42. Mattison OR, Thomford PJ: The mechanisms of action of reproductive toxicants. In Working P (ed): Toxicology of the Male and Female Reproductive Systems. New York, Hemisphere Publishing, 1989, pp 101-129 43. Mattison OH, Shiromizu K, Nightingale MS: Oocyte destruction by polycyclic aromatic hydrocarbons. Am J Ind Med 4:191, 1983 44. Mattison OR, Shiromizu K, Nightingale MS: The role of metabolic activation in gonadal

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Address reprint requests to Donald R. Mattison Department of Obstetrics and Cynecology University of Arkansas for Medical Sciences 4301 West Markham Little Rock, AR 72205