Sexual differentiation of reproductive neuroendocrine function in sheep

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1 Reviews of Reproduction (1998) 3, Sexual differentiation of reproductive neuroendocrine function in sheep Ruth I. Wood 1 and Douglas L. Foster 2 1 Departments of Obstetrics and Gynecology and Biology, Yale University School of Medicine, New Haven, CT , USA; and 2 Reproductive Sciences Program, and Departments of Obstetrics and Gynecology, and Biology, University of Michigan, Ann Arbor, MI , USA In many species, the timing of puberty is different in males and s. This does not simply reflect differences in the time course of activation of the testes and ovaries. Rather, sex differences in pubertal onset reside within brain mechanisms controlling GnRH secretion, as exemplified by studies conducted in sheep. Exposure of sheep fetuses to testicular steroids alters the timing of puberty, principally by reducing photoperiod responsiveness. This is manifest as an early increase in LH secretion in males or in s exposed experimentally to testosterone before birth. Steroids also act on non-photoperiodic mechanisms to abolish the preovulatory gonadotrophin surge. In view of these multiple organizational actions of steroids to control postnatal gonadotrophin secretion, it is becoming clear that there are many critical periods of brain development for organizing the GnRH neurosecretory system, and that these may be sensitive to different testosterone metabolites. Although GnRH neurones are not sexually dimorphic with respect to number, distribution or gross morphology, fundamental questions remain as to how steroids exert their effects at the cell through actions on GnRH afferents. Teleologically, these early sex-specific changes in mechanisms timing puberty maximize the chance that reproductive activity will ultimately be successful in each sex. Puberty, the transition from a sexually immature state to one of full reproductive activity, is timed by signals acting in the brain to increase activity of the hypothalamo pituitary axis which stimulates the production of mature gametes. There are profound sex differences in the timing of puberty in many species. This occurs by sexual differentiation of the neuroendocrine control of pituitary gonadotrophin secretion. Studies using sheep as an experimental model have defined some of the factors responsible for this developmental difference in reproductive neuroendocrine function. We have studied the prenatal organizers (prenatal steroids), postnatal cues (growth and photoperiod responses) and the effectors (GnRH neurones) that time the pubertal process. Our experimental strategy has been to conduct complementary anatomical and physiological investigations to link form and function in the sexual differentiation of reproductive neuroendocrine function. Reproductive strategies in males and s To ensure that sexual activity will ultimately be successful, males and s respond to cues from both their internal and external environments. Mating is timed so that young are born when somatic conditions are appropriate and seasonal conditions are favourable. However, the differences in reproductive strategies between males and s can often be as wide as those between different species (Bronson, 1989). These differences arise because the time course of gametogenesis and the metabolic investments in reproduction can be quite different in the two sexes Journals of Reproduction and Fertility /98 $12.5 The typical mammal gives birth to relatively few offspring during her lifespan, but expends considerable energy in their growth and early development (for review, see Bronson, 1989). Thus, most of her reproductive effort is invested after mating. The metabolic demands of pregnancy and lactation are considerable, and can compromise the chances of survival of the if begun at an inappropriate time. However, the gametic cycle of s is relatively short, and only a few days are required to develop a preovulatory follicle. Together, these factors encourage the immature to delay sexual maturation until conditions are optimal. After puberty, the typical adult living in natural conditions may have only a few fertile cycles in 1 year. For much of the year, she is reproductively suppressed as a result of pregnancy and lactation, seasonal anovulation or, possibly, inadequate food intake. Nonetheless, once internal and external cues are favourable, reproductive activity can be initiated rapidly. The male uses an entirely different strategy to time sexual activity. The typical polygynous male produces large numbers of offspring, but does little to ensure their survival. Thus, most of the energy for his reproductive effort is expended before mating, primarily in competition for s (Bronson, 1985). Even though the energetic demands for production of spermatozoa and seminal fluid are comparatively small, spermatogenesis takes weeks to months to complete (Ortavant, 1958). Thus, the male benefits by beginning and maintaining spermatogenesis despite inadequate nutrition and adverse environmental conditions (Bronson, 1989). In sheep, puberty in males begins about 2 weeks before it does in s (Fig. 1b). Spring-born male lambs begin

2 Sexual differentiation of reproductive neuroendocrine function 131 reproductive development in midsummer at about 1 weeks of age, as evidenced by the onset of spermatogenic cycles (Ortavant, 1958; Claypool and Foster, 199), whereas lambs remain prepubertal until mid-autumn, when they first ovulate (at approximately 3 weeks; Foster and Ryan, 1979; Claypool and Foster, 199). Admittedly, this comparison alone is of little value. The endpoints of sexual maturation in males and s of any species are difficult to equate because of differences in production of male and gametes (continuous production of spermatozoa in males compared with cyclic production of ova in s). Nevertheless, the underlying neuroendocrine changes that lead to puberty in the two sexes are similar. Neuroendocrine sequence for puberty and its timing in male and lambs The increase in the secretion of gonadotrophins, particularly LH, from the pituitary drives the transition from sexual immaturity to reproductive competence in both sexes (Fig. 1c) (Kosut et al., 1997). Before puberty, the brain, which controls the secretion of LH via GnRH, is highly sensitive to the inhibitory effects of steroids from the developing gonads. Hence, the frequency of GnRH pulses remains low. At puberty, in response to cues from the internal (growth) and external environment (photoperiod), the brain becomes less sensitive to the inhibitory effects of gonadal steroids. The frequency of GnRH secretion increases which, in turn, increases the pulsatile secretion of LH to drive follicular growth and spermatogenesis. In addition, in s, increasing concentrations of circulating oestradiol at puberty trigger the first preovulatory LH surge (Fig. 1d) (for review, see Foster, 1994). In males, the preovulatory LH surge is neither present nor inducible (Karsch and Foster, 1975) because increments of oestradiol cannot produce a surge of GnRH (Herbosa et al., 1996). Neither the gonad nor the anterior pituitary of sexually immature sheep limit fertility, since both are capable of functioning well before the time of puberty. The concept that centrally regulated hypogonadotrophism limits puberty is based upon several considerations: (1) the potential to produce high frequency pulses of LH is attained at a few weeks of age; (2) the gonad is capable of a sustained increase in steroidogenesis early in postnatal development; and (3) in s, the gonadotrophin surge mechanism can respond to the stimulatory feedback actions of oestradiol within a few weeks after birth (for review, see Foster, 1994). In sheep, the gonadostat hypothesis is applicable to puberty in both sexes. The gonadostat refers to the sensitivity of the GnRH neurosecretory system to inhibitory feedback of gonadal steroids. According to this hypothesis, production of gonadotrophins in the prepubertal individual is low because feedback sensitivity is high. This sensitivity to gonadal steroids decreases during sexual maturation and, as a consequence, the secretion of GnRH increases to stimulate LH secretion. This important concept has been demonstrated in an experimental model in which the gonads are removed and a constant steroid feedback signal is provided by Silastic implant. Oestradiol is used in this experimental model because both male and lambs have a similar degree of responsiveness to oestradiol or testosterone with regard to inhibition of LH pulse frequency (Olster and Daylength (h) Testes size (cm 2 ) LH (ng ml 1 ) GnRH (pg min 1 ) MAR (a) (b) JUN SEP DEC Breeding season Testicular growth and spermatogenic cycles begin Ovulatory cycles begin Age (weeks) 1 (d) Surge No surge (c) Time (h) Time (h) % Cycling Foster, 1986), and because oestradiol plays an important role in inhibitory steroid feedback not only in ewes, but also in rams (Schanbacher, 1984). The gonadectomized, oestradiol-treated model is useful for comparing neuroendocrine sexual maturity in males and s because the same variable, the pubertal LH increase, is measured in both sexes (Fig. 1c). The model eliminates the differences between males and s in steroid production, gametogenesis and sexual behaviour. Before puberty, LH pulse frequency remains low in the gonadectomized lamb of either sex bearing a steroid implant (male: Olster and Foster, 1986; : Foster et al., 1986). Eventually, high frequency LH pulses can be expressed in the presence 5 LH (ng ml 1 ) Fig. 1. Timing of puberty in spring-born male and lambs in relation to age and photoperiod. (a) Annual photoperiod curve in Ann Arbor, Michigan. (b) Indices of pubertal maturation from gonad-intact males and s. (c) Circulating LH concentrations from gonadectomized oestradiol-treated male ( ) and ( ) lambs. (d) Circulating concentrations of GnRH and LH during 48 h from a representative lamb (left) and male lamb (right) in response to a surge-inducing dose of oestradiol. Redrawn from Claypool and Foster, 199 (a, b) and data from Kosut et al., 1997 (c) and Herbosa et al., 1996 (d).

3 132 R. I. Wood and D. L. Foster of inhibitory steroid feedback, and they occur in synchrony with gonadal maturation in the intact male or (Fig. 1b) (Claypool and Foster, 199). This neuroendocrine sexual maturity is dependent on the sex of the lamb. In s, the decrease in sensitivity leading to an increase in LH occurs at approximately weeks of age (Foster and Ryan, 1979; Claypool and Foster, 199). By contrast, in males, the duration of hypersensitivity lasts only half this time, and by about 1 15 weeks of age, high frequency LH pulses are expressed (Olster and Foster, 1986). Mechanisms for sexual differentiation of reproductive neuroendocrine function Both the prenatal organization and postnatal activation of the reproductive neuroendocrine system have been examined to understand how reproductive neuroendocrine function becomes sexually differentiated. Three approaches have been used: (1) evaluation of prenatal gonadal steroids in the postnatal control of gonadotrophin secretion to understand how sex differences in neuroendocrine function arise; (2) investigation of sex differences in responsiveness to internal and external cues that activate the reproductive neuroendocrine system postnatally; and (3) comparison of GnRH secretion and neuroanatomy in males, s, and prenatally androgenized s to uncover an anatomical basis for sex differences in reproductive neuroendocrine function. Masculinization of the brain It is now axiomatic that testicular steroid hormones are the principal effectors of differentiation towards a masculine phenotype (reviewed in Goy and McEwen, 198). Sex steroids have actions in the developing brain distinct from their effects at puberty and beyond. Unlike the transient activational effects of steroids in adulthood, exposure to these steroids during a critical period in early development produces permanent, organizational changes in central nervous system structure and function (see Gorski, 1985). The size of the sexually dimorphic nucleus of the preoptic area in rats described by Gorski et al. (1978) is perhaps the best-known example of organizational changes in the central nervous system (CNS). Functional changes associated with organizational effects of gonadal steroids include masculinization of sexual behaviour and defeminization of the LH surge (MacLusky and Naftolin, 1981). Within the broad critical period for sexual differentiation, each sexually dimorphic trait is presumed to have an individual period of susceptibility to steroids (that is, individual critical periods) (Clarke et al., 1976; Christensen and Gorski, 1978; Gorski and Jacobson, 1981). These critical periods occur prenatally in sheep and other species with long gestation periods (for example, primates and guinea-pigs); in contrast, the brain becomes sexually differentiated mostly in the first few days after birth in rats and other species with short gestation periods (Goy and McEwen, 198). As an experimental strategy to study how steroid hormones alter reproductive neuroendocrine function, males can be feminized by depriving them of gonadal steroids during the critical period or, alternatively, s can be masculinized with exogenous steroids. Both approaches have been used in rodents, in which the critical period is largely postnatal, to study sexual differentiation of sexual behaviour and the LH surge (MacLusky and Naftolin, 1981). In sheep, castration of males at 3 days of gestation is technically not feasible (145 days is term). However, steroid administration to s prenatally has been used to reveal which sexually differentiated traits are determined by the actions of prenatal androgens, when the critical periods are for sexual differentiation, and which steroids are involved (Short, 1974; Clarke et al., 1976; Wilson and Tarttelin, 1978). Prenatal androgens advance neuroendocrine puberty As initially determined by Short (1974), and later refined by Clarke et al. (1976), sexual differentiation in lambs occurs from approximately 3 to 1 days of the 145 day gestation. Females born to testosterone-treated mothers display masculinization of the external genitalia, urination posture and behaviour, as well as impaired reproductive function indicated by lack of oestrus, sporadic ovulation, and a blunted LH surge (Short, 1974; Clarke et al., 1976; Clarke and Scaramuzzi, 1978). However, in gonadintact androgenized lambs, the time of sexual maturity is not readily identifiable. The LH surge mechanism is often muted, and ovulation is sporadic. This problem has been overcome through use of the steroid-clamp model described above (data illustrated in Fig. 1c). Pulsatile LH secretion in gonadectomized, oestradiol-treated androgenized s provides a measure of neuroendocrine sexual maturation unobscured by steroid effects on the LH surge or at the ovary. Exposure of lambs to testosterone from 3 to 86 days of gestation by weekly maternal injection of 2 mg testosterone cypionate caused masculinization of the external genitalia and defeminization of the LH surge (Fig. 2b)(Wood et al., 1991a). Exposure to this same dose at a later stage of gestation (between day 89 and day 135 of gestation) was without effect (Herbosa and Foster, 1996). These results confirmed the findings of the earlier Fig. 2. Effects of androgen treatment in utero on external genitalia, neuroendocrine sexual maturation, and the LH surge in lambs. Column 1: schematic of type of androgen treatment. Column 2: schematic sagittal section through the hind-quarters of representative male, and androgenized lambs illustrating the position of the penis, scrotum or vulva. Column 3: LH concentrations in gonadectomized oestradiol-treated lambs from samples collected twice per week. Column 4: LH response to follicular phase concentrations of oestradiol over 6 h. Androgenization occurred during the entire critical period (Wood et al., 1991a). (a) Normal male lamb. (b) Female lamb treated with high dose testosterone from day 3 to day 86 of gestation. (c) Normal lamb. (d,e) Effect of stage of development (Wood et al., 1995). (d) Female lamb treated with testosterone from day 3 to day 5 of gestation. (e) Female lamb treated with testosterone from day 65 to day 85 of gestation. (f h) Effect of amount of testosterone (Kosut et al., 1997). (f) Female lamb treated with high dose of testosterone (2 mg week 1 ). (g) Female lamb treated with medium dose of testosterone (8 mg week 1 ). (h) Female lamb treated with low dose of testosterone (32 mg week 1 ). (i) Effect of type of steroid: androgenized s exposed to high doses of dihydrotestosterone (DHT) throughout the critical period (K. Masek, R. Wood, and D. Foster, unpublished).

4 Sexual differentiation of reproductive neuroendocrine function 133 (a) (b) Treatment None Testosterone External genitalia Normal male 2 1 Tonic LH Surge 4 2 LH (ng ml 1 ) (c) None Normal (d) (e) (f) (g) Early Late High Medium LH (ng ml 1 ) LH (ng ml 1 ) (h) Low n=2 n=4 (i) DHT 3 9 Gestation (days) Age (weeks) Time (h) 4 2 LH (ng ml 1 )

5 134 R. I. Wood and D. L. Foster studies by Iain Clarke and Roger Short (Short, 1974; Clarke et al., 1976). Of great interest was the pattern of LH secretion in these gonadectomized, oestradiol-treated androgenized lambs. It was clear that prenatal androgens advanced the onset of neuroendocrine sexual maturation to 1 weeks of age, much the same as occurs in males (Fig. 2a). This provided, for the first time, an explanation for the earlier activation of the gonads in males, namely that the pubertal LH increase occurs at a young age in male lambs because the tonic mode of LH secretion is masculinized by testosterone in utero. Moreover, this observation set the stage for subsequent investigations of how prenatal steroids might masculinize and defeminize the control of the tonic and surge modes of gonadotrophin secretion. Differential masculinization of tonic and surge LH In view of the emerging concept that the critical period is not a single entity, it is important to understand if the two modes of gonadotrophin secretion have unique requirements for androgenization. This has been studied with respect to the stage of development, and the amount and type (androgen versus oestrogen) of steroid exposure (Fig. 2). Influence of stage of development. The timing of steroid exposure contributes to the type and degree of masculinization (Fig. 2d,e). Androgen administration to mothers for 2 days during the early part of the critical period (days 3 5) was compared with that during the latter portion of the critical period (days 65 85; Wood et al., 1995). These two androgen treatments had markedly different effects on the external genitalia, similar to those of previous studies (Short, 1974; Clarke et al., 1976). Females exposed to testosterone during early gestation possessed a penis and empty scrotum, whereas those treated during later development were normal. Despite clear differences in their outward appearance, reproductive neuroendocrine function was similar in the two groups of androgenized s. In both groups, tonic LH secretion increased at 18 2 weeks of age, well before it does in normal s, but much later than it does in males. Moreover, these androgenized s were capable of responding to oestradiol stimulation with an increase in LH secretion. However, compared with normal s, the LH surges in androgenized s were delayed by several hours. These results suggest that it is possible to advance the pubertal increase in tonic LH significantly without abolishing the LH surge. The broad implication is that complete defeminization of the LH surge requires a longer exposure to testosterone compared with that required to masculinize tonic LH. The finding that the timing of the surge was altered, but not completely abolished, by prenatal testosterone raises the interesting possibility that certain types of idiopathic infertility could be explained by a developmentally induced subtle delay in synchrony of the preovulatory surge mechanism with follicular development. Perhaps in such a condition, the GnRH surge mechanism has reduced sensitivity to oestradiol stimulatory feedback, and the surge is delayed until an advanced stage of the follicular phase, resulting in ovulation of a postmature follicle. Influence of amount of steroid. In addition to the stage of development and duration of androgen exposure, the amount of testosterone in utero is an important determinant of postnatal masculinization (Fig. 2f h). Therefore, we determined the effects of increasing doses of testosterone on the androgenization of tonic and surge modes of LH secretion (Kosut et al., 1997). Pregnant ewes received weekly injections of 2 mg, 8 mg or 32 mg testosterone cypionate for the duration of the now established critical period (days 3 9 of gestation). After birth, masculinization of the external genitalia was dose dependent such that high doses of testosterone cypionate completely masculinized the genitalia, whereas low-dose androgen treatment had no effects. Intermediate doses of androgen produced moderate genital masculinization. Masculinization of tonic LH secretion also exhibited a dose response effect. Tonic LH in lambs treated prenatally with high doses of androgens increased at 1 weeks, much like it did in normal males. Medium-dose androgenized s exhibited an increase in tonic LH at 16 weeks, well before that in control s. Tonic LH remained at basal concentrations in low-dose androgenized s until 25 weeks, similar to the case in control s (27 weeks). However, none of the androgenized s treated with high or medium doses of testosterone, and only two of six s exposed to low dose androgens, produced a surge of LH in response to exogenous oestradiol. This finding suggests that the developmental mechanisms organizing the surge system are more sensitive than tonic secretion to the amount of testosterone exposure in utero. Influence of type of steroid (androgen versus oestrogen). A surprising feature of CNS sexual differentiation is that, in many species, oestradiol is a more potent masculinizing agent than is testosterone (MacLusky and Naftolin, 1981) (Fig. 2i). Presumably, this is because testosterone is converted to oestrogen locally by the aromatase enzyme. Testosterone can also be reduced to dihydrotestosterone (DHT) by 5α-reductase. Because DHT cannot be metabolized to oestrogen, it is considered to have a pure androgenic action. DHT is without effect on sexual differentiation in most rodents (Whalen and Rezek, 1974; Korenbrot et al., 1975). However, there is growing evidence from species with long gestation periods, including guinea-pigs and monkeys, that androgens per se contribute to masculinization of sexual behaviour and neuroendocrine function (Goldfoot and van der Werff ten Borsch, 1975; K. Wallen, personal communication), although oestrogens also play a role (Hines et al., 1987; Goy and Deputte, 1996). Indeed, preliminary information from an ongoing study in which DHT was used to masculinize lambs indicates that the pubertal LH increase is advanced to 8 1 weeks of age, but that the LH surge system is still present (K. Masek, V. Mehta, R. Wood, and D. Foster, unpublished). If this finding holds true, it raises the interesting possibility that masculinization of the tonic mode of GnRH secretion occurs through an androgen-dependent mechanism, whereas defeminization of the surge mode is via oestrogen produced from testosterone by local aromatization. Together, the foregoing studies reveal that masculinization of the tonic mode of GnRH secretion and defeminization of the surge mode of GnRH secretion are differentially sensitive to characteristics of the prenatal steroid signal. In particular, it appears that masculinization of tonic GnRH secretion leading to early puberty is achieved by a relatively brief, concentrated exposure to androgens before birth, whereas the LH surge is

6 Sexual differentiation of reproductive neuroendocrine function 135 defeminized by low concentrations of oestrogen (converted from testosterone) over a longer period of fetal development. The important implication is that the mechanisms controlling tonic pulsatile LH release are distinct from those that determine the positive feedback response to oestradiol. However, the neural elements controlling tonic and surge secretion of GnRH have not yet been identified. Internal and external factors timing puberty In all species, internal cues are used to ensure that the appropriate growth has been achieved to time puberty. In most species, external cues relating to seasonal or social factors are used as well. Growth is clearly important in sheep, in which daylength is also used to time the pubertal increase in tonic LH secretion to the appropriate season (Foster, 1994). Because male lambs begin sexual maturation about 2 weeks before the developing, they do so at a lower body weight and during a different season. Thus, the growth or photoperiod, or both, requirements timing puberty must be different in male and sheep. One way in which prenatal exposure to steroids times neuroendocrine sexual maturation is by setting the postnatal responsiveness to these internal and external factors. Growth The general consideration is that information about growth and metabolism is conveyed by neural and blood-borne signals to the CNS. If these somatic signals indicate that growth is inadequate, such that reproduction will place excessive metabolic demands upon the young lamb, sexual maturation is inhibited. This is comparable to anorexia nervosa in humans (Foster et al., in press). High frequency tonic LH secretion begins only once an adequate stage of growth is achieved. Identification of the exact substances used as signals, the location of their sensors, and the pathways by which this information is sent to the GnRH neurosecretory system are as yet unknown but are of considerable interest (Foster et al., in press). It is unlikely that either these signals or their pathways are sexually differentiated, but until any of them have been determined, this will not be possible to assess. In theory, the sensitivity to metabolic signals could differ between the two sexes in sheep, as has been demonstrated in mice (Hamilton and Bronson, 1985). However, a comparative study of growth restriction and refeeding in male and lambs suggests an equivalent neuroendocrine sensitivity to metabolic cues (Wood et al., 1991b). This finding indicates that sex differences in growth and metabolic requirements for puberty are not the major determinants of the earlier onset of puberty in males. Photoperiod Profound sex differences in sensitivity to external cues have been identified. After achieving the appropriate stage of body growth, a young sheep must then ensure that reproductive activity is initiated during the optimal season of the year. Only when both requirements for puberty are satisfied can sexual maturity be exhibited (Foster et al., 1986). In Michigan, spring-born lambs (Suffolk breed) attain puberty in the autumn (at approximately 3 weeks of age), shortly after the start of the adult breeding season (see Foster et al., 1989). In contrast, young males begin spermatogenesis at approximately 1 15 weeks of age, regardless of season (Courot et al., 1975). The seasonal factor timing puberty in s is photoperiod, more specifically when daylength decreases from long to short days (Yellon and Foster, 1985), as occurs outdoors after the summer solstice (Fig. 1a,b). In contrast, male lambs are relatively insensitive to changes in photoperiod (Courot et al., 1975). In fact, the pubertal increase in gonadotrophins in spring-born males begins during the long days of summer (Olster and Foster, 1986), a photoperiod that is normally inhibitory to puberty in the (Ebling and Foster, 1988). Sex differences in photoperiodism have also been described for ferrets (see Wood et al., 1991c). However, in other species, males and s are either both sensitive to photoperiod to time puberty (for example, Djungarian hamsters), or are both non-responsive to changes in daylength until after sexual maturation (for example, Syrian hamsters). This sex difference in the reproductive response to photoperiod in young lambs is the result of the organizing action of testicular androgens during fetal development, as evidenced by the timing of sexual maturity in androgenized lambs maintained under controlled photoperiods (natural simulated or reverse natural simulated (Fig. 3a,b)). Under a natural simulated photoperiod, tonic LH secretion increases in gonadectomized, oestradiol-treated males at 7 weeks, when days are lengthening. In normal s treated similarly, sexual maturity occurs at 27 weeks when daylengths decrease. The reverse photoperiod has no effect on males, but it delays the expression of sexual maturity in s beyond 32 weeks of age. Prenatal treatment of s with androgens masculinizes the reproductive response to photoperiod and renders them insensitive to the inhibitory effects of lengthening photoperiods; in these androgenized s under a reversed natural simulated photoperiod, LH increases at 7 weeks, a similar time to the LH increase in males (Herbosa et al., 1995). Androgens masculinize this reproductive response to photoperiod only during the critical period, as evidenced by the age of sexual maturity in lambs treated with testosterone during the majority of the critical period (days 3 76) or later in gestation (days ) when reared under a photoperiod (constant long days) normally inhibitory to s (Herbosa and Foster, 1996) (Fig. 3c). By the end of the study (4 weeks), only one of seven normal s had a pubertal increase in LH. In contrast, all males increased LH secretion at an average of 7 weeks. In s androgenized during the critical period, a sustained increase in LH occurred at 17 weeks, later than in normal males, but well before the increase in control s. It is likely that exposure for the entire known critical period (days 3 9), instead of only 75% of it (days 3 76), would have resulted in a complete masculinization of the photoperiod response and the pubertal increase at the same age as that for males (Fig. 3a,b). Testosterone treatment after the critical period during the last third of gestation had little, if any, effect on tonic LH secretion; only three of eight s had sustained increases in LH. These s were able to produce an LH surge in response to oestradiol stimulation, while those exposed during the critical period were not. Although it has become apparent that steroids contribute to the timing of puberty in male and lambs through

7 136 R. I. Wood and D. L. Foster Photoperiod (h) (a) (b) (c) (n = 7) (n = 8) (n = 8) (n = 8) (n = 7) (n = 6) 11 1 (n = 8) (n = 8) Early Late (n = 8) (n = 6) (n = 7) Age (weeks) Fig. 3. Effect of photoperiod on neuroendocrine sexual maturation in lambs. The beginning of the pubertal increase in tonic LH secretion in each gonadectomized, oestradiol-treated lamb ( ). (a) In natural simulated photoperiod, the pubertal LH increase in normal s occurs during decreasing daylengths whereas, in androgenized s, this occurs at the same time as in normal males. (b) Reverse natural simulated photoperiod delays the pubertal increase in LH in s, but not in males or in androgenized s. (c) Constant long days delay sexual maturation in normal s and in androgenized s exposed to androgens after the critical period for sexual differentiation (late gestation, days ). For s masculinized during the early part of the critical period (days 3 76), the pubertal increase in LH is intermediate between that of normal males and s. Redrawn from Herbosa et al., 1995 (a, b) and Herbosa and Foster, 1996 (c). sexual differentiation of photoperiod requirements, it is not known where in the brain such differences are manifest. This is because there is a paucity of information about neural mechanisms through which seasonal information is communicated to the reproductive neuroendocrine system in any species. To use changes in daylength, both young and adult sheep acquire a photoperiod history built on changes in the circadian pattern of pineal melatonin (Foster et al., 1986). However, it is clear that sex differences in photoperiodism are related to interpretation of the rhythm of melatonin, and not to the secretion of melatonin itself. The melatonin rhythms of male and lambs are not different (Claypool et al., 1989), despite the fact that males and s respond to changes in daylength differentially, each according to their own particular photoperiod requirements for sexual maturation. Functional and anatomical effects of prenatal androgens on GnRH neurones Neuroanatomical investigations that complement the foregoing physiological studies have begun to assess where and how steroids are perceived by the brain to induce an early reduction in responsiveness to steroid feedback and to defeminize the LH surge. Sexual differentiation of neural circuits controlling reproductive neuroendocrine function will undoubtedly include the steroid-responsive neurones, which transduce steroid cues, and GnRH neurones, which are the principal effectors of gonadotrophin control. However, information on the groups of steroid receptor-containing neurones that control neuroendocrine function, and their links to the GnRH neurones is limited. Therefore, studies to date have focused on the pattern of GnRH secretion and the GnRH neurone itself. GnRH secretion It has long been assumed that the inability of male sheep to produce an LH surge stems from the absence of a stimulatory feedback mechanism and lack of a GnRH surge. Until recently, this hypothesis could not be tested directly because it was not possible to measure GnRH secretion in males in response to acute oestradiol stimulation. Methods to access the pituitary portal vasculature have been developed (Caraty et al., 1994) and adapted for use in the lamb (Herbosa et al., 1996), which allowed us to test the hypothesis that a sex difference in the pattern of oestradiol-stimulated GnRH secretion is established by gonadal steroids during prenatal development. Pituitary portal GnRH was monitored in males, s, and androgenized s. As expected, in each normal, there was an unambiguous increase in GnRH after treatment with oestradiol, and each surge of GnRH was accompanied by a concomitant increase in LH (see Fig. 1d left for representative ). In marked contrast, in males, there was no sustained increase in GnRH in response to oestradiol (Fig. 1d right). Treatment with testosterone in utero clearly modified the pattern of GnRH secretion to the extent that no LH surges were produced in androgenized s, suggesting that the hypothalamic drive that stimulates the LH surge is not functional in male sheep and that this mechanism is organized prenatally by testosterone, probably through conversion to oestradiol.

8 Sexual differentiation of reproductive neuroendocrine function 137 Fig. 4. GnRH neuroanatomy in the lamb. Rostrocaudal distribution of GnRH neurones ( ) from (a) the diagonal band of Broca, (b) preoptic area, (c) anterior hypothalamus, (d) the mediobasal hypothalamus of a sheep fetus during the middle of gestation. (e) Electron micrograph of a GnRH neurone. Arrowheads indicate afferent synapses, which occur most often on small spine-like protuberances from the soma and proximal dendrites. Insets illustrate selected synapses at higher magnification. AHA: anterior hypothalamus; alac: anterior limb of the anterior commissure; fx: fornix; LS: lateral septum; ME: median eminence; MPOA: medial preoptic area; MS: medial septum; mt: mammillothalamic tract; NDB: nucleus of the diagonal band of Broca; oc: optic chiasm; pit: anterior pituitary; PVN: paraventricular nucleus; SON: supraoptic nucleus; st: stria terminalis; VMH: ventromedial hypothalamic nucleus. Redrawn from Wood et al., 1996 (a d); (e) data from Kim et al., Scale bar represents 25 nm.

9 138 R. I. Wood and D. L. Foster Male Female Fetal testis T DHT E 2 Surge Steroid-responsive neurones Photoperiod GnRH Medial preoptic area Growth Median eminence LH Anterior pituitary 16 Spring Autumn 1 Photoperiod (h light per day) 4 2 LH (ng ml 1 ) Age (weeks) 3 Fig. 5. Sex differences in reproductive neuroendocrine function in lambs: current hypotheses. Steroids from the fetal testis act on steroid-responsive neurones in the male brain to reduce synaptic to GnRH neurones. Specifically, oestrogens eliminate stimulatory connections responsible for triggering the preovulatory GnRH surge, while androgens (and possibly oestrogens as well) reduce inhibitory synapses that determine postnatal photoperiod responsiveness. However, lambs of both sexes are sensitive to signals about growth and metabolism. GnRH neuroanatomy Distribution and gross morphology. GnRH neurones are few in number and are scattered widely throughout the preoptic area, the diagonal band of Broca and, to a lesser extent, in the anterior hypothalamus (Fig. 4 a d). Few cell bodies are present in the arcuate nucleus or the ventromedial nucleus close to the site of GnRH release at the median eminence. Unlike those of most other species, the GnRH neurones of sheep are frequently multipolar cells with extensive branching processes. There are no sex differences in the number, location, or gross morphology of GnRH neurones between male and sheep fetuses (Wood et al., 1992), as is true of ferrets, rats and monkeys. Moreover, the anatomy of the GnRH system at mid-gestation is similar to that of adult ewes (Lehman et al., 1986). Oestradiol-induced activation. Prenatal steroids prevent activation of GnRH neurones in response to oestradiol, perhaps

10 Sexual differentiation of reproductive neuroendocrine function 139 by uncoupling the GnRH neurone from stimulatory inputs of oestrogen-sensitive neurones. This was tested by comparing Fos colocalization with GnRH in the brains of males, s, and androgenized s in response to a surge-inducing dose of oestradiol (Wood et al., 1996). Fos, the protein product of the immediate-early gene, c-fos, can be detected immunocytochemically in the cell nucleus of individual neurones after oestradiol stimulation, and is presumed to reflect increased activity of those neurones. Sex differences in the activation of GnRH neurones in rats and ferrets have been described using Fos as a marker of neural activity (Hoffman et al., 199; Lambert et al., 1992). In lambs, oestradiol induced Fos immunostaining throughout steroid-responsive limbic regions in all lambs, regardless of their exposure to prenatal androgens. However, the colocalization of Fos with GnRH was highly sexually dimorphic. In brains of control lambs after the peak of the oestrogen-induced LH surge, Fos was expressed in 67% of GnRH neurones, similar to an earlier report by Moenter et al. (1993). In contrast, in males and androgenized s, only 2% of GnRH neurones were Fos-positive. Synaptic input. Although no evidence for marked sex differences in GnRH neuroanatomy is apparent using light microscopy, some differences appear at the ultrastructural level (Fig. 4). An attractive hypothesis to explain the actions of gonadal steroids in sexual differentiation of the CNS is that steroids have growth-promoting effects on neurones, including stimulation of synaptogenesis. Oestradiol promotes synapse formation in limbic regions of the rat brain (see Garcia-Segura et al., 1994), and GnRH neurones of rats receive nearly twice as much synaptic input as those of males (Chen et al., 199). GnRH neurones of adult sheep during the breeding season also possess a greater number of synapses compared with ewes during anoestrus (Xiong et al., 1997). In sheep, there is a sex difference in synaptic input to GnRH neurones, which is under the influence of prenatal steroids (Kim et al., 1996). GnRH neurones from gonadectomized, postpubertal lambs received approximately twice as many contacts as those of male lambs or androgenized s. Because GnRH neurones themselves do not have receptors for gonadal steroids (Shivers et al., 1983), it is presumed that steroids acting on GnRH afferents promote this connectivity. At present, it is not known whether the additional complement of GnRH afferents in s is excitatory or inhibitory, nor whether these synapses contribute preferentially to oestradiol stimulation of the GnRH surge or to photoperiodic regulation of tonic GnRH secretion. Conclusions These studies in sheep add to an increasing literature describing the process through which the developing individual differentiates towards a masculine or feminine phenotype. According to current understanding, puberty in males depends only on growth. This enables the young male to activate testicular maturation at an early age to begin to compete for s. The, regardless of how fast she grows, is ultimately reliant on daylength cues to initiate a 5 month pregnancy such that birth and lactation occur in the spring. Whereas the growth requirements for puberty may be similar in males and s, photoperiodic inhibition of gonadotrophin secretion in s masks early expression of sexual maturation. An important feature of the sheep model is that exposure to androgens during a critical period for sexual differentiation of the brain alters the timing of the expression of sexual maturity by decreasing the reliance upon photoperiod (Fig. 5). In addition, and most likely through a separate mechanism involving an oestrogen pathway, prenatal testosterone alters the timing and function of the GnRH surge mechanism. Potential cellular mechanisms for prenatal steroid action include altering connectivity of the neural elements that control GnRH release. The authors thank Juanita Pelt for assisting with the illustrations; Katherine Masek for use of preliminary data on DHT; Shepard Kosut, Vikas Mehta, Katherine Reese, Cristina Herbosa, Deborah Olster, and Lee Claypool, all of whom during their training and work with the sheep model at Michigan helped to develop the concepts described; Michael Lehman and Sarah Newman, who assisted with neuroanatomy; and Iain Clarke and Roger Short for the original observations that set the direction of the research. This work was supported by grants from the United States Department of Agriculture ( , , , , ) and from the National Institutes of Health for core facilities (P3-HD-18258) and training (T32-HD-748). References Key references are identified by asterisks. Bronson FH (1985) Mammalian reproduction: an ecological perspective Biology of Reproduction Bronson FH (1989) Mammalian Reproductive Biology The University of Chicago Press, Chicago Caraty A, Locatelli A, Moenter SM and Karsch FJ (1994) Sampling of hypophyseal portal blood of conscious sheep for direct monitoring of hypothalamic neurosecretory substances. In Pulsatility in Neuroendocrine Systems, Methods in Neuroscience Vol. 2 pp Ed. JE Levine. 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