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Reproductive Sciences Program and Department of Biology (K.S.M., D.L.F.), and Department of Obstetrics and Gynecology and Biology (D.L.F.), University of Michigan, Ann Arbor, Michigan 48109-0404; and the Departments of Obstetrics and Gynecology and Molecular, Cellular, and Developmental Biology, Yale University (R.I.W.), New Haven, Connecticut 06520-8063
Address all correspondence and requests for reprints to: Dr. Douglas L. Foster, Room 1101, 300 North Ingalls Building, Ann Arbor, Michigan 48109-0404. E-mail: dlfoster{at}umich.edu
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Abstract |
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Introduction |
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The reproductive neuroendocrine system in sheep is responsive to prenatal organization and subsequent activation by gonadal steroids at puberty. In females, estrogen triggers a massive surge of LH secretion to cause ovulation, but in males, this mechanism is neither present nor inducible (3). This sex difference in pituitary LH secretion is determined by sex differences in the neural elements controlling the release of GnRH from the hypothalamus (4, 5). A marked sex difference also exists in the control of tonic GnRH secretion at puberty in the sheep. This mechanism, characterized by changes in the frequency of pulsatile LH release, is responsible for the pronounced difference in the timing of puberty in the lamb (6). The pubertal increase in tonic LH secretion, indicative of reduced responsiveness to steroid negative feedback, begins at 10 weeks of age in males (7). In females, LH concentrations remain at baseline until 30 weeks of age. In our earlier studies, testosterone treatment of female lambs in utero induced an early increase in tonic LH and abolished the LH surge (8, 9, 10, 11). The contributions of androgens and estrogens to brain sexual differentiation in sheep have not been determined.
In the present study we assessed the relative importance of aromatization in the sexual differentiation of the tonic and surge modes of LH secretion and expression of sexual behavior. We exposed developing female lambs to the nonaromatizable androgen, dihydrotestosterone (DHT). Whereas testosterone can be converted to estrogen, DHT cannot and is considered to have a "pure" androgenic effect. Our findings reveal that distinct requirements exist for differentiation of the two major types of reproductive hormone controls in the sheep. Aromatization is not essential for prenatal masculinization of the postnatal pattern of tonic, pulsatile LH secretion. However, conversion to estrogen is required for sexual differentiation of the LH surge and sexual behavior.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, top). Ewes received 800,
400, or 200 mg DHT/week for 9 weeks until day 90 (term is 145–150
days). The time of DHT treatment in utero spanned the
critical period for sexual differentiation of the reproductive
neuroendocrine system. Previous studies by Short (12) and Clarke
et al. (13) and our laboratory (8, 9, 10, 11) have determined that
exposure to testosterone during this period masculinizes developing
female lambs.
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, bottom).
Tonic LH secretion
We used a well characterized model routinely used in our
laboratory to assess whether aromatization is required for
differentiation of the tonic mode of LH secretion. The timing of the
pubertal rise in LH was determined in gonadectomized lambs chronically
treated with estradiol. With the initiation of sexual maturity, a
marked decrease in sensitivity to the inhibitory feedback action of
estradiol contributes to the sustained rise in circulating
gonadotropins (7). To standardize the sex steroid environment during
sexual maturation, endogenous steroids were removed via gonadectomy,
and an estradiol-containing capsule was implanted sc to provide
constant steroid feedback. The pronounced pubertal increase in LH in
ovariectomized estradiol-treated females coincides with the initiation
of ovulations and estrous cycles in ovary-intact females (14).
Estradiol is an important feedback hormone regulating LH secretion
before puberty in the male lamb as well (6, 15, 16). Males have a
similar degree of responsiveness to estradiol and testosterone with
regard to the regulation of LH pulse frequency (15). Moreover, the
increase in tonic LH secretion in estradiol-treated castrated male
lambs coincides with the onset of testicular growth and
spermatogenic cycles in gonad-intact males (6).
Gonadectomy and steroid replacement were performed at 4 weeks of age.
Untreated and androgenized females were ovariectomized under anesthesia
(atropine, 0.2 mg/kg; ketamine, 20 mg/kg; Rompun, 0.1 mg/kg) via a
midline abdominal incision. Testes were removed under local lidocaine
anesthesia. At gonadectomy, the external genitalia of each lamb were
described and measured. To produce chronic steroid feedback, we used
SILASTIC brand tubing (od, 0.46 cm; id, 0.34 cm; Dow Corning Corp., Midland, MI) with a 30-mm packed column of crystalline
17ß-estradiol (Sigma Chemical Co.), which was sealed
with SILASTIC brand adhesive type A (Dow Corning Corp.) at
each end. Before their sc insertion, the implants were soaked overnight
in water to prevent a postimplantation surge in steroid release (17).
This implant maintains circulating estradiol in both male and female
lambs at physiological levels (
3–5 pg/ml) (6). Throughout the
40-week study, blood samples (5 ml) were collected via jugular
venipuncture twice weekly and allowed to coagulate. Serum was collected
and frozen until assayed for LH by RIA. Serum LH concentrations were
analyzed in 25- to 200-µl volumes using modifications (18, 19) of a
RIA described by Niswender et al. (20). Assay sensitivity
(defined as 2 SD above maximum binding) was 0.73 ng/ml for
200 ml serum (15 assays), expressed relative to NIH LH-S12. Intraassay
coefficients of variance, based on two quality control pools known to
bind the standard curve at 20% and 50%, averaged 6.6% and 6.4%,
respectively. Interassay coefficients of variance averaged 12.32% and
7.9% respectively.
The criterion established previously in our laboratory (21) was used to evaluate the timing of reproductive neuroendocrine maturation in the gonadectomized, estradiol-treated lambs. The onset of the pubertal rise, reflecting the decrease in sensitivity to inhibitory steroid feedback, was defined as the first time when circulating LH exceeded 1 ng/ml for 3 consecutive weeks (first of six consecutive LH samples). All between-group comparisons were made by ANOVA using Scheffe’s F test.
LH surge
The influence of prenatal DHT on the positive feedback system
was studied using progesterone and estradiol in an artificial
follicular phase model previously described (22, 23). The LH surge
system was evaluated at 39 weeks of age, after the pubertal increase in
tonic LH had occurred in all androgenized females and in control males
and females. To induce robust LH surges, we pretreated the lambs with
progesterone. Chronic estradiol implants were removed, and 1 week
later, circulating estrogen and progesterone were increased to luteal
phase concentrations. Progesterone was delivered via two controlled
internal drug-releasing (CIDR) devices inserted sc (300 mg
progesterone/CIDR; Interag, Hamilton, New Zealand). Estrogen
replacement was performed using a small (10-mm) SILASTIC brand implant
sc. The CIDRs were removed after 10 days, and 24 h later, all
lambs received four 30-mm SILASTIC brand estradiol implants sc to
produce high follicular phase levels of circulating estradiol (12–15
pg/ml) (24). LH was measured in blood samples collected every 2 h
for 6 h before and 48 h after the beginning of estrogen
stimulation. A LH surge was defined as LH values exceeding twice the
average preestradiol baseline for a minimum of 6 h (three
consecutive samples).
Sexual behavior
All lambs were tested for male and female sexual behavior during
evaluation of the surge system. In adult females, sexual receptivity
accompanies the LH surge (25). However, estrogen also induces
significant expression of masculine sexual behavior in rams (26).
Measures of female sexual behavior were based on the methods described
by Clarke and Scaramuzzi (27); those for masculine behavior were from
those reported by Crichton et al. (26). To determine the
onset of female receptive behavior, a sexually experienced ram with
marking paint was housed with the lambs (one ram per pen of eight
lambs) during estradiol stimulation. During the next 48 h, lambs
were inspected for marks every 2 h. In addition, to evaluate the
quality of female sexual behavior after 24 h of estradiol
stimulation, each lamb was paired with a ram for 10 min. Proceptive
(head turns, fanning, nudging), receptive (standing), and agonistic
behaviors were recorded by an observer. Afterward, masculine sexual
behavior was evaluated in each lamb for 10 min during exposure to an
estrous female. Courtship (nudging, kicking, ano-genital
investigation), copulatory (mounting), and agonistic behaviors were
recorded. Differences between groups were evaluated by Fisher’s exact
probability test, adjusting for multiple comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), similar to the effects of
testosterone in utero (8, 9, 10, 11). DHT-treated females were
easily identified at birth by the absence of testes in the scrotum. As
observed during ovariectomy at 4 weeks of age, DHT-treated females had
ovaries and a uterus in the normal intraabdominal position. However,
the uterus merged with the urethra to exit through a penis
externally.
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). As sensitivity to steroid
feedback becomes reduced, tonic LH rises (14). In control males (n
= 7), this began at 6.7 ± 0.5 weeks of age (mean ±
SEM). In females (n = 9), the onset of increased LH
secretion was significantly later, at 29.1 ± 0.8 weeks
(P < 0.05). The control of tonic LH secretion was
masculinized to a similar degree in all three groups of DHT-treated
females. For females exposed to 800 mg (n = 5), 400 mg (n =
4), and 200 mg (n = 5) DHT/week, the pubertal rise in tonic LH
began at 10.8 ± 1.8, 9.9 ± 3.0, and 7.1 ± 2.2 weeks
of age, respectively. This was the same age as that for normal males,
but it was significantly earlier than that for control females
(P < 0.05).
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). In contrast, no
sustained increase in LH occurred in any control male. However, unlike
the masculinization of tonic LH, prenatal DHT failed to defeminize the
LH surge. Estrogen induced LH surges in all but one DHT-treated female.
Moreover, the duration and latency of the LH surge in DHT-treated
females were not significantly different from those in control females.
Even in the two females that did not produce a LH surge (one control
and one 200 mg DHT-treated female), LH levels increased transiently
after estrogen stimulation.
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). By
15 h after estrogen treatment, 8 of 9 control females were marked.
The remaining female (no. 740) failed to show sexual behavior at any
time during the study, although she did produce a robust surge of LH.
In contrast, none of the control males was marked during the 48-h
observation period (P > 0.05). Only 2 of 12
DHT-treated females were marked during the first 24 h of estrogen
treatment; 1 DHT-treated female from the 800 mg group was marked at
10 h, and 1 was marked at 16 h (P < 0.05
compared with control females).
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Estrogen stimulation elicited only modest masculine sexual behavior,
even in control male lambs (Fig. 6).
During 10 min of exposure to a receptive female, 5 of 6 control males
investigated the ewe’s anogenital region, although none exhibited
tongue flicking or flehmen. Half of the males showed foreleg kicking
and nudging. However, only 2 of 6 control males mounted the stimulus
female. In control females, 2 of 9 investigated the stimulus ewe’s
ano-genital region (P < 0.05 relative to males), and
only 1 displayed kicking or nudging. None of control females mounted
the stimulus ewe, and 2 expressed agonistic behavior. Likewise,
estradiol stimulation failed to induce substantial masculine sexual
behavior in DHT-treated females. Only 1 of 12 showed anogenital
investigation (P < 0.05 compared with males), and 2
displayed foreleg kicking.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aromatization of testicular androgens is critical for the rat, in which sexual differentiation takes place largely during the early postnatal period (1, 2). However, as evident by the results of the present study, conversion of testosterone to estrogen is not necessarily required for brain differentiation in all species. Perhaps the aromatization hypothesis is largely valid for species in which sexual differentiation develops postnatally. However, androgens may play a greater role in sexual differentiation of long gestation species, where masculinization occurs prenatally. In guinea pigs and rhesus monkeys, two other long gestation species, androgens contribute to masculinization of sexual behavior (28). Thus, in sheep, in which sexual differentiation of the control of LH secretion is complete long before the time of birth (12, 13), one would expect that some component of the reproductive neuroendocrine system would be sensitive to the organizing action of androgens as well. Our findings reveal that this is indeed the case.
It appears that prenatal androgen may alter the timing of the pubertal increase in GnRH secretion largely through uncoupling the reproductive neuroendocrine system in the developing male lamb from regulation by photoperiod cues. The male lamb is relatively insensitive to photoperiod; he begins pubertal development at 10 weeks of age regardless of day length (29). By contrast, the control of tonic LH secretion and the timing of the pubertal LH rise are exquisitely sensitive to photoperiod in the female lamb (7). Spring-born females increase tonic LH only when day lengths decrease after the summer solstice. Thus, prenatal steroid treatments that advance puberty in female lambs must reduce the reliance on photoperiodic cues (10). It is noteworthy that the surge system is not responsive to changes in day length, and exogenous estrogen can trigger a surge of LH at any time of year (30). If prenatal androgens act selectively on photoperiod mechanisms, it is not surprising that DHT-treated females retain the ability to produce a LH surge.
Early studies of sexual differentiation classified steroid effects as organizational (permanent structural changes during development) and activational (transient effects in adulthood) (1). However, these two steroid effects may not be entirely independent. Instead, we hypothesize that for any sexually dimorphic trait, the steroids responsible for organizational changes are likely to be the same hormones that cause activation in adulthood. In this regard, nonaromatizable androgens cannot induce the LH surge in adult female sheep (31, 32). Likewise, nonaromatizable androgens cannot defeminize the LH surge in developing females of any species examined (2) including sheep, as evidenced from the present study. However, both DHT and estrogen can inhibit tonic LH secretion in adult male and female sheep (33). The present study demonstrates that tonic LH secretion is sensitive to the organizational effects of DHT, and we further predict that prenatal estrogens will masculinize tonic LH secretion in addition to defeminizing the LH surge. Indeed, both androgen and estrogen may be required for full masculinization of the control of tonic GnRH secretion. If so, this may explain the low amplitude of the LH rise in DHT-treated females from the present study.
To probe the hormonal requirements for sexual differentiation, an
alternate approach to masculinization of the female is feminization of
the developing male with steroid receptor blockers or enzyme
inhibitors. Although administering exogenous hormones to females
determines which steroids can masculinize the developing fetus,
blocking the action of an endogenous hormone presumably reveals which
steroids masculinize the brain under normal circumstances. This
approach has been used in guinea pigs (34, 35). Prenatal exposure to a
5
-reductase inhibitor did not feminize reproductive neuroendocrine
function, although it did block masculinization of the external
genitalia (34). According to our hypothesis that estrogen acts on both
tonic and surge LH, we would anticipate similar results in sheep. What
is more surprising is that an aromatase blocker also failed to feminize
GnRH secretion in male guinea pigs, although pituitary responsiveness
to GnRH was reduced (35). Perhaps in guinea pigs, testosterone is
required for defeminization of the preovulatory surge. However, it is
also possible that the aromatase blocker did not eliminate estrogen
formation. These results highlight the difficulties of blocking
endogenous metabolism of testosterone. As DHT and estradiol are each
more potent than testosterone alone, even a small amount of residual
enzyme action can be sufficient for masculinization to occur.
The links between organizational and activational effects of steroids may also explain the effects of prenatal DHT on sexual behavior. Testosterone stimulation of male sexual behavior in sheep (26), as in many rodent species (36), is mediated by aromatization to estrogen. DHT replacement to castrated males has little effect. Likewise, prenatal testosterone stimulates masculine sexual behavior and inhibits female mating behavior in androgenized female lambs (27, 37, 38). In the present study, prenatal exposure to DHT failed to induce significant male sexual behavior in response to estradiol stimulation. This finding accords with the effects of DHT in adult male sheep. Although prenatal DHT does contribute to behavioral masculinization in guinea pigs (39), aromatization is not essential for sexual behavior in this species (40). It should be noted that induction of male mating behavior generally requires a longer duration of steroid exposure (26). Hence, even control males in this study were unable to express robust sexual activity. With regard to female sexual behavior, aromatization of testosterone to estrogen is required for defeminization in a variety of species (41), including sheep (present study). It is possible that DHT-treated females may be less attractive to rams, thereby accounting for the reduced incidence of marking in the present study. However, rams were willing to engage in courtship and copulatory behavior during individual pairing, including scenting urine from the penis of DHT-treated females.
The underlying sex dimorphisms in the sheep brain that determine sex differences in reproductive neuroendocrine function and sexual behavior remain unclear. Ultimately, the differential steroid responsiveness of tonic and surge modes of LH secretion is probably determined by the differential distribution of androgen and estrogen receptors in the presynaptic neurons that control GnRH release. Although the present study did not measure GnRH directly, we have used LH secretion to make inferences about the pattern of hypothalamic GnRH release. It is reasonable to do so, at least in sheep. With simultaneous measurement of LH in jugular blood and GnRH from the hypothalamo-hypophyseal portal system, a pulse of GnRH in portal blood precedes each pulse of LH in the systemic circulation (42). Moreover, pituitary responsiveness to exogenous GnRH is not sexually dimorphic in sheep (43). These data suggest that masculinization of reproductive neuroendocrine function in sheep is largely through steroid effects on brain, particularly on the GnRH neuronal system. The gross morphologies of the GnRH system in males and females are similar (44). To date, a sex difference in the number of synapses on GnRH neurons has been noted (45), but the phenotype of the presynaptic neurons is not known. Although this distribution can be altered by administration of testosterone to females in utero, the relative importance of androgens and estrogens remains to be determined.
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Acknowledgments |
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Footnotes |
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Received December 9, 1998.
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References |
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-reductase activity in sexual differentiation of the guinea pig.
Neuroendocrinology 49:324–330[CrossRef][Medline]
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