Differential sensitivity of mounting and lordosis control systems to early androgen treatment in male and female hamsters.

The effects of early testosterone propionate (TP) treatment on the adult sexual behavior of hamsters were investigated in two experiments. In Expt. I, male and female pups were injected with oil vehicle or 1, 5, 10, 50, 100, or 250 μg of TP 24 hr after birth. In Expt. II, males and females received either oil or 10 μg of TP on the day of birth (Day 1), Day 3, Day 5, Day 7, or Day 9. At 70 days of age all animals were gonadectomized and 10 days later tested for lordosis behavior after estrogen and progesterone priming. One week after the test for female behavior all females began receiving 500 μg of TP each day and were tested for mounting and intromission behavior three times at 10 day intervals. Lordosis behavior was inhibited by as little as 5 μg of TP given 24 hr after birth. In males this dose produced the maximal effect, but in females increasing dosages resulted in a proportional decrease in lordosis duration. One μg of TP neonatally facilitated later mounting and intromission behavior in females and 250 μg of TP was no more effective than 1 μg. Lordosis duration was inhibited in females by 10 μg of TP on either Day 1 or 3, however, mounts and intromissions were facilitated by TP treatment on Day 1, 3, 5 or 7. These experiments demonstrate that the mechanisms mediating masculine behavior are more sensitive to neonatal TP treatment than are the mechanisms mediating lordosis behavior.     

Effects of early neonatal thyroxine treatment on development of the thyroid and adrenal axes in rats

Neonatal male rats were treated with intraperitoneal (IP) injections of thyroxine on postnatal days 1 to 4. Plasma triiodothyronine (T₃), thyroxine (T₄), and corticosteroids were determined on days 5, 10, 13, 16, 20, 30, and 60 of age, and all animals were weighed weekly. Neonatal thyroxine treatment resulted in reduced body weight throughout the entire test period, decreased T₃ and T₄ until day 60 of age, and elevated corticosteroids on days 5 to 13 of age. These results demonstrate that early neonatal T₄ treatment affects the ontogeny of both thyroid and adrenocortical hormones, and suggest that these developmental changes should be considered when evaluating the consequences of T₄ treatment during the early neonatal period.     

Effects of septal lesions on behavioral sensitivity of female rats to gonadal hormones

Spayed female rats were given bilateral septal lesions or a sham operation and 3 wk later tested for hormone-induced female sexual behavior. When primed with 0.5, 1.0, or 2.0 μg of estradiol benzoate (EB) per day for 3 days and tested for lordosis behavior on the fourth day, animals with septal lesions showed a positive dose-related increase in mean lordosis quotient (LQ), whereas control animals showed a low mean LQ for all doses of EB. After priming with a low dose of EB (0.5 μg/day for 3 days), progesterone administration prior to behavior testing on day 4 produced a comparable facilitation in LQ for both septal-lesioned and sham-operated animals. When treated for 3 days with either 50 or 150 μg of testosterone propionate (TP) and given progesterone prior to behavior testing on day 4, female rats with septal lesions showed a higher mean LQ than sham-operated rats. Thus, septal lesions increase the behavioral sensitivity of female rats to both EB and TP as measured by female sexual behavior, but do not appear to alter the responsiveness of animals to progesterone.     

Effect of intermittent injections of gonadotrophin releasing hormone at various frequencies on the release of luteinizing hormone and ovulation in dairy cows

Gonadotrophin releasing hormone (GnRH, 5 μg every 4 h) was administered to six dairy cows between days 5 and 10 post-partum and the release of luteinizing hormone (LH) and the onset of ovulation were determined. LH was measured using a specific radioimmunoassay and the occurrence of ovulation was assessed from changes in the concentration of progesterone in milk. Treatment with GnRH resulted in a median time of first ovulation of 17.0 days after calving. This was less (P < 0.05) than that observed for control cows (21.5 days, n = 7). Determinations of plasma LH concentrations over an 8-h period on days 6 and 10 post-partum indicated that there was a tendency for GnRH-treated cows to have higher levels of LH on these days. The 5 μg dose of GnRH did not repeatably induce a release of LH between days 6 and 10. Endogenous pulsatile release of LH did, however, increase in frequency from 3.18 pulses per 8 h on day 6 to 5.18 pulses per 8 h on day 14 post-partum (P < 0.01).In a second experiment groups of 20 cows were treated with either 5 μg GnRH every 4 h or 15 μg GnRH every 12 h from days 5 to 10 post-partum. Seventeen untreated cows served as controls. The median times to first ovulation were 27.0 days for the control cows, 22.5 days for those cows treated with 5 μg GnRH every 4 h and 17.0 days for cows treated with 15 μg every 12 h. The latter treatment significantly advanced the time of first ovulation (P < 0.05) relative to controls. This difference had, however, disappeared by the time of the second and third ovulations. Primiparous cows ovulated later (P < 0.01) than the pluriparous cows in the group treated with 5 μg GnRH every 4 h. This was a major reason for the lack of effect of this treatment. Some treated cows were blood sampled at frequent intervals on day 8 to evaluate the LH responses to GnRH injections. The administration of 5 μg GnRH on day 8 did not elicit a pulse of LH which could be distinguished from endogenous pulsatile secretion at this time. The dose of 15 μg on this day did, however, elicit a more defined pulse on some, but not all, occasions.The injection of a small dose of GnRH twice a day from day 5 to day 10 after calving, therefore, advanced the time of first ovulation in dairy cows by 10 days.     

Effects of thyroxine and triiodothyroacetic acid on cerebral myelin lipids

Neonatal rats, thyroidectomized at one day of age, received either no replacement therapy or daily subcutaneous injections of 100 μg/kg body weight of thyroxine (T₄) or equal molar amounts of triiodothyroacetic acid (T₃AC) from day 6 to day 26. Myelin lipid was isolated from the rat cerebrums and the cholesterol, cerebroside and total phospholipid content were determined. Cerebral myelin lipid weights were decreased in the neonatally thyroidectomized and T₄ treated rats. The myelin lipid weights of the T₃AC treated rats were not different from those treated with T₄. The lipid composition remained unchanged in the T₃AC, T₄, and thyroidectomized groups when compared with the normal rats. These results indicate that T₃AC and T₄ administration have similar effects on CNS myelination in brains of neonatally thyroidectomized rats.     

Alterations in mammary gland development following neonatal exposure to estradiol,transforming growth factor α, and estrogen receptor antagonist ICI 182,780

High fetal/early postnatal levels of estrogen increase breast cancer risk, but the mechanisms remain unknown. Growth factors, such as transforming growth factor α (TGFα), may participate as secondary modifiers in this process. We characterized a modulatory role of early postnatal exposure to 17β-estradiol (E₂) on the developing mammary gland morphology by treating intact female CD-1 mice with physiological doses of E₂ (2–4 μg), human recombinant TGFα (4 μg), or an estrogen receptor (ER) antagonist ICI 182,780 (20 μg) during postnatal days 1–3. Early postnatal exposure of E₂ stimulated mammary ductal growth by days 25 and 35, but by day 50 this was inhibited. The level of differentiation from terminal end buds (TEBs) to the lobulo-alveolar units (LAUs) also was reduced by day 50. The number of TEBs was increased throughout most of the development in the female mice exposed to E₂ during early life. An exposure to TGFα or ICI 182,780 between postnatal days 1 and 3 stimulated ductal growth, formation of TEBs, and the differentiation of mammary epithelial structures. ICI 182,80 treatment also caused hyperplastic lobular-like structures in 54-day-old females. Thus, neonatal exposure to TGFα and ICI 182,780 induced both similar (increase in TEBs) and different (increase/decrease in lobulo-alveolar differentiation) developmental changes in the mouse mammary gland, when compared with an exposure to E₂. A unique feature of the postnatal E₂ treatment was that it inhibited ductal migration by days 50–54. Our data suggest than an exposure to E₂ on postnatal days 1–3, possibly combined with secondary epigenetic alterations, leads to various changes within the developing mammary tree. These changes may be potential prerequisites for mammary tumorigenesis. J. Cell. Physiol. 170:279–289, 1997. © 1997 Wiley-Liss, Inc.     

Mounting behavior and lordosis behavior in castrated male rats treated with testosterone propionate, or with estradiol benzoate or dihydrotestosterone in combination with testosterone propinonate.

Treatment of prepuberally castrated male rats with testosterone propionate (TP, 50, 200, 500, or 1000 μg for 30 days) in adulthood stimulated the display of both mounting behavior and lordosis behavior. No correlation between mounting and lordosis behavior could be detected at any TP dose level. Treatment of prepuberally castrated male rats with either 1 μg estradiol benzoate (EB) or 500 μg dihydrotestosterone (DHT) for 60 days stimulated the display of mounting behavior in three of eight and four of eight rats, respectively. Treatment with 200 μg TP for the last 30 days of rats receiving either EB or DHT for 60 days resulted in an abrupt onset on mounting behavior as compared to rats treated with oil for 60 days. These results show additive effects of EB or DHT and TP upon mounting behavior by male rats and are interpreted as a support for the suggestion that testosterone to estrogen as well as testosterone to DHT conversion may be involved in the mechanism whereby testosterone activates the mounting behavior of castrated rats.     

Early androgen treatment and male and female sexual behavior in mice

To investigate the role of neonatal androgen stimulation in the development of the potential for masculine and feminine sexual behavior in the mouse, different groups of mice were hormonally manipulated early in life. One group of female mice was administered testosterone propionate (TP) within 24 hr of birth; a second group of females was given a control injection of oil on the day of birth; a third group of females received an injection of TP on the 10th day after birth. A group of males received a control injection of oil on the day of birth. All mice were gonadectomized at about 30 days of age. At 60 days of age, mice were injected with estrogen and progesterone and tested for sexual receptivity; several weeks later all mice were injected with TP and tested for male sexual behavior. Female behavior: Females given oil at birth and females given TP on the 10th day after birth showed high levels of sexual receptivity as adults following estrogen-progesterone treatment. Females given TP on the day of birth, and male mice, rarely exhibited lordosis following estrogen-progesterone treatment. Male behavior: Most mice, regardless of genetic sex or neonatal treatment, mounted in adulthood following administration of exogenous androgen. There was little difference in mounting frequency between groups, suggesting that exogenous or endogenous androgen stimulation of the neonatal mouse does not facilitate adult mounting behavior. These data for the mouse are in essential agreement with existing data for the rat, and indicate that sexual behavioral differentiation induced by androgen stimulation in infancy is best characterized as an inhibition of the potential to display feminine sexual behavior in adulthood.     

Effect of prolonged administration of small doses of LH-RH and LH-RH analogue [D-Ser (But)6] Des Gly-NH210 ethylamide on the release of LH and induction of ovulation in mid-anoestrous ewes

A natural process of LH release and induction of ovulation in anoestrous ewes was simulated by prolonged administration of small doses of LH-RH and its analogue [D-Ser(But)⁶] Des Gly-NH₂¹⁰ ethylamide. In the first series of experiments on 40 Merino ewes infusions of LH-RH were made into the maxillaris interna artery for 6 consecutive days for 6 h each day. Total doses of 24.0, 26.0, 28.0 and 32.0 μg per animal of varying and progressively increasing daily quantities of the hormone were administered in groups I, II, III and IV, respectively. In group V the animals were infused with a total dose of 28.0 μg LH-RH and injected additionally i.m. with 3.0 μg 17β-oestradiol on days 4 and 5 of the infusion of LH-RH. Ovulation did not occur earlier than days 4, 5 and 6 after the beginning of infusions. The highest number of positive reactions occurred in group IV ($\text{8}\text{10}$) and in group V ($\text{7}\text{8}$ animals). The pattern of LH peaks in general was correlated with the time of ovulations. The LH concentrations of the preovulatory peaks in experimental ewes were mostly lower than those in naturally ovulating animals. The corpora lutea were functional during the first 7 days after ovulation.In the second series of experiments on 26 Merino ewes the LH-RH analogue [D-Ser -(But)⁶] Des Gly-NH₂¹⁰ ethylamide was injected i.m. or i.a. for 6 consecutive days. Total doses of 15.5, 9.5 and 7.5 μg of the analogue per animal, administered at varying and progressively increasing daily doses in respective groups, induced several surges of LH in the same individuals for 2 or even 3 consecutive days. Corpora lutea and degenerating follicles in the form of cysts were found in the ovaries of animals of these groups. Very small daily doses ranging from 0.1 μg administered during the first 3 days, to 1.5 μg on day 5 of the treatment, released one surge of LH on day 5 of the treatment in all individuals with peaks ranging from 30.0 to 58.0 ng/ml and induction of ovulation with almost normal luteal function. On the basis of these experiments it is suggested that the evaluation of the effect of active substance (LH-RH or its analogue), its suitability and application of rightly chosen doses to induce the full physiological process of ovulation should be based not only on the release of LH and luteal function but also on tests of the ability of the released ovum to undergo fertilization and its further development.     

Development of corneal transparency in embryonic chick: influence of exogenous thyroxine and thiouracil on structure, water and electrolyte content.

Thiouracil and thyroxine (T₄) were injected onto the chick chorioallantois at various developmental stages to study their effects on corneal cellularity, dehydration and structure. Corneas were excised 2–9 days after treatment for histological examination and for analyses of water content, sodium concentration [Na⁺] and potassium concentration [K⁺]. Untreated chick corneas showed that water content and [Na⁺] decreased with advancing embryonic age, while [K⁺] increased up to stage 42 and then rapidly declined. Corneas from embryos injected with 10 mg thiouracil at stage 36 had a significantly reduced [K⁺] at stages 40 and 42. Corneas at stages 40, 42 and 45 had a significantly elevated water content when compared with controls. Injection of 15 μg of T₄ prior to stage 36 or at or after stage 40 did not produce significant changes in corneal water and ionic content compared with controls; injection of 15 μg of T₄ during the cell proliferation period of corneal development (stages 36–40) produced a significant increase in [K⁺]. Similar results were obtained in corneas from embryos injected with 1 μg of T₄. Total corneal thickness was increased in thiouracil treated corneas, and decreased in T₄ treated corneas. Epithelial growth was markedly decreased with thiouracil treatment, while T₄ had little effect. It is likely that thiouracil treatment decreases cell division in the cornea, and prevents formation of the epithelial barrier, whereas T₄ accelerates these processes.     

Regulation of formation of covalently bound flavins in liver and cerebrum by thyroid hormones.

The effects of thyroxine (T₄) and triiodothyronine (T₃) treatment upon the formation of [2-¹⁴C]flavins bound covalently to tissue proteins in liver and cerebrum were measured 1 h after a subcutaneous injection of [2-¹⁴C]riboflavin in male rats of different ages. In livers of rats of ages 2, 3, and 12 months, T₄ (100 μg/100 g body wt) and T₃ (25 μg/100 g body wt) in daily intraperitoneal doses for 7 days each increased incorporation into covalently bound flavins 50% above that in saline-treated controls. In newborn rats, T₄ in doses of 10 μg/rat for 7 days increased incorporation similarly to that in adults. In adult rats doses of T₃ from 2.5 to 25 μg/100 g body wt were nearly as effective as larger doses of T₃ and T₄ in increasing the formation of covalently bound flavins in liver. In cerebra of newborn rats, T₄ was ineffective in increasing the formation of covalently bound flavins. However, in cerebra of rats of ages 2, 3, and 12 months, both T₃ and T₄ consistently increased the formation of covalently bound flavins. Doses of T₃ from 2.5 to 25 μg/100 g body wt produced significant increases. These findings are of interest in view of our previous demonstration that the formation of flavin adenine dinucleotide, the major tissue flavin, is not increased in rat brain even by massive doses of thyroid hormones. The present results indicate that the formation of the fraction of flavins bound covalently to tissue proteins differs from the usual pattern of brain metabolism of adult rats in being subject to control by thyroid hormones.     

Effects of neonatal septal lesions on reproductive behavior of male and female rats

The purpose of this study was to examine the effects of neonatally placed septal lesions (SL) in male, female, and androgenized female rats on reproductive behavior. Animals were castrated as adults and tested for both feminine and masculine sexual behavior. After treatment with estradiol benzoate (EB) alone (2 μg daily for 3 days), only the females with SL which had not been given testosterone propionate (TP) neonatally showed a facilitation of lordosis behavior. Following EB (2 μg for 3 days) plus 0.5 mg progesterone (P), both the lesioned and the sham-operated female groups showed an increase in the display of lordosis in either hormonal condition. All animals were given a pretest for masculine sexual behavior and tested on Days 4, 7, 11, and 15 of daily TP treatment (150 μg/day). There was no effect of the neonatally placed SL on masculine sexual behavior in female rats or in female rats androgenized with 30 μg TP. However, lesioned females treated neonatally with 1 mg TP showed a marginal enhancement of masculine sexual behavior. Male rats given SL neonatally showed a marked enhancement of masculine sexual behavior compared to that of controls. These results suggest that, depending on the neonatal hormone environment, SL selectively increase behavioral sensitivity to hormones. Although neonatally lesioned females show behavioral responses similar to females given SL as adults, male rats given SL neonatally are unique in that they show enhanced masculine sexual behavior whereas males lesioned as adults do not.     

Effects of cortisol administration on development of the thyroid and adrenal axes in rats

Intraperitoneal (IP) injections of cortisol were administered to neonatal male rats on postnatal days 1 to 4. Plasma triiodothyronine (T₃), thyroxine (T₄) and corticosteroids were determined on days 5, 10, 13, 16, 20, 30 and 60 of age, and all animals were weighed weekly. Neonatal cortisol treatment resulted in depressed body weight gain and transient depression in plasma T₄. These results indicate that while body weight is significantly affected by cortisol treatment ontogenic patterns of plasma T₃, T₄ and corticosteroids develop normally.     

Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T₄) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T₄ was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T₄ also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T₄ and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development.     

Regulation of fetuin A gene expression in the neonatal pig liver

Fetuin A (also known as α2-Heremans–Schmid glycoprotein) is a protein primarily expressed by the liver and secreted into the blood. Previous studies have suggested that plasma concentrations of fetuin A are elevated with impaired growth rate in swine. The present study was designed to examine the relationship of porcine fetuin A with growth rate in the pig and to also elucidate the regulation of fetuin A expression by examining the hormonal and cytokine regulation of fetuin A mRNA abundance in hepatocytes prepared from suckling piglets. Quantitative real-time PCR assay was used to quantify the number of fetuin A mRNA molecules/molecule cyclophilin mRNA. Total RNA was isolated from liver of three different groups of pigs to assess changes in mRNA abundance of fetuin A: normal piglets at day 1, day 7 day 21 or 6 months of age (n=6 for each age); runt and control piglets at day 1 of age (n=4); slow growing and normal growing piglets at 21 days of age (n=8). Following birth, fetuin A gene expression increased from day 1 and 7 of age (P<0.05), and then declined at 21 days of age (P<0.05), with a much greater decline to 6 months of age (P<0.01). Fetuin A mRNA abundance was higher in runt pigs v. their normal birth weight littermates (P<0.05). Similarly, fetuin A gene expression was higher in livers of pigs that were born at a normal weight but that grew much slower than littermates with the same birth weight (P<0.05). Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h to permit examination of the influences of hormones, cytokines and redox modifiers on fetuin A mRNA abundance. Fetuin A gene expression was enhanced by glucagon, T3 and resveratrol (P<0.05). Growth hormone, cytokines (interleukin6, tumor necrosis factor-α) and antioxidants (N-acetylcysteine, quercertin) reduced fetuin A mRNA abundance (P<0.05). A role for fetuin A in postnatal development is suggested by the differences in fetuin A mRNA abundance between runt piglets or slow growing piglets and their normal growing sized littermates. The hepatocyte experiments suggest multiple hormones and cytokines may contribute to the regulation of fetuin A during early growth of the pig.     

Thyroid hormones are required for daily rhythms of plasma corticosterone and prolactin concentration

Daily patterns of plasma corticosterone (B) and prolactin (PRL) concentration were measured in female rats which were intact (INTACT), thyroidectomized (THYREX), or thyroidectomized and given thyroxine (T₄) replacement (THYREX + T₄) (20 μg T₄/day). Bimodal daily rhythms of plasma B were present in INTACT rats and THYREX + T₄ rats. However, no plasma B rhythm was detectable in THYREX rats. THYREX + T₄ rats, which had greater mean T₄ concentrations than INTACT rats (6.0 μg/dl vs. 3.5 μg/dl), had a plasma B rhythm of greater amplitude than INTACT rats. Plasma PRL rhythms were detectable in INTACT and THYREX + T₄ rats, but not in THYREX rats. INTACT rats had a single peak in plasma PRL, whereas three plasma PRL peaks were present in THYREX + T₄ rats. It was concluded that thyroid hormones are required for the expression of plasma B and PRL rhythms and that the levels of thyroid hormones can alter the amplitude of the B rhythm and the shape of the PRL rhythm.     

Estrogen plus androgen induced mounting in adult female hamsters

This study demonstrated that the combined administration of estrogens and androgens activates the display of mounting by female hamsters. Forty-nine ovariectomized hamsters were injected daily with either estradiol benzoate (EB, N = 8); dihydrotestosterone propionate (DHTP, N = 7); testosterone propionate (TP, N = 6); androstenedione (AD, N = 9); EB plus DHTP (N = 10); or estrone plus DHTP (E₁ + DHTP, N = 9). All androgens were administered at a dose of 1 mg per day for the first 24 days and at a dose of 2 mg per day for the last 14 days. The EB dose was 6 μg per day and the E₁ dose was 100 μg per day. Females were tested for male behavior once a week starting on Day 10 of injections and for female behavior on Day 39.One hundred percent of EB + DHTP treated females; 67% of the E₁ + DHTP treated females; 55% of the AD treated females; 33% of the TP treated females; 29% of the DHTP treated females; and none of the EB treated females mounted during at least one test. Only one of the E₁ + DHTP treated females showed the intromission pattern; otherwise most females which mounted displayed the intromission pattern. The median number of days preceding the onset of mounting ranged from 21 to 31 days and did not differ among treatment groups.     

Cerebral myelinogenesis in theSnell dwarf mouse: Stimulatory effects of GH and T4 restricted to the first 20 days of postnatal life

We attempted to define the critical time period during early postnatal life when GH and T₄ are essential for myelination. We administered bGH and T₄ toSnell dwarf mice during the first and second 20 days after birth. Positive results were obtained only when hormones were given during the first 20 days of postnatal life. We observed a distinct increase in brain weight, DNA content, CNPase activity and a remarkably increased level of spontaneous locomotion activity with a diurnal periodicity. Morphological observations of brain sections stained for myelin basic protein (MBP) correlated the biochemical findings. The later administration of hormones was ineffective. Our interpretation is that the administration of exogenous hormones led to increased myelinogenesis through their stimulatory effects on glial proliferation, as evidenced by the increase in cerebral DNA content.     

A single injection of 17 beta-estradiol facilitates sexual behavior in castrated male mice

Sexual behavior was assessed in castrated adult CD-1 male mice given exogenous steroids under various treatment regimens. Castrated mice maintained on 20 μg testosterone (T) daily for 1 week, but given 250 μg testosterone propionate (TP) on the day of testing showed higher levels of copulatory activity than intact mice or the males receiving an additional dose of 20 μg T on the test day, although plasma testosterone levels were not different at the time of behavioral testing. Castrated males given 50, 125, or 250 μg TP for 1 week including the day of testing showed higher levels of sexual behavior than males receiving the same doses of TP only once, on the test day. A single injection of 17β-estradiol (E₂) completely restored the male copulatory pattern, including ejaculation, in castrated mice under every condition examined. Testosterone and dihydrotestosterone (DHT) were less effective than E₂, as was the combination of E₂ and DHT. The relative efficacy of a single dose of T, DHT, and E₂ plus DHT was dependent upon factors such as the delay between steroid administration and testing, as well as whether or not the castrated mice received androgen replacement prior to testing. Estradiol benzoate (E₂B) was not capable of restoring sexual behavior in castrated mice in this study. The comparison of results obtained with TP, T, E₂, and E₂B suggests that an appreciable, but not necessarily sustained, elevation of E₂ levels in the brain may be critical in the facilitation of male copulatory behavior in mice.