Neuronal cell death during sexual differentiation and lateralisation of vocal communication.

A rodent analogy has been established to investigate the neural mechanisms occurring during sexual differentiation and lateralization. A sexually dimorphic hypothalamic nucleus (SDApc) is closely associated with a stereotyped, courtship vocalisation in male gerbils. Stereological analysis of SDApc cytoarchitecture reveals that neuron number and nuclear volume are asymmetric in male adults. Strikingly, neuron number on the left side of the SDApc correlates significantly with the rate of the courtship call in males. Exogenous testosterone treatment in female neonates masculinises and lateralises SDApc structure and function. Neuronal programmed cell death (apoptosis), manifested in SDApcs of neonates, is more frequent in females. Significantly, apoptosis in males is lateralised, as revealed by lateral asymmetry of neuron number at postnatal day 16. It is concluded that neuroendocrine-dependent, sexual differentiation and lateralization are concurrent and influenced by apoptotic mechanisms. It is suggested that apoptosis is the result of a genetically-driven device, inherent in postmitotic, undifferentiated cells which may have recently migrated into the SDApc. The genomic mechanism inducing lateralised apoptosis is apparently activated only neonatally in males.

Laterally asymmetrical cell number in a sexually dimorphic nucleus in the gerbil hypothalamus is correlated with vocal emission rates.

A lateralized relationship between the total volume of a discrete area in a hypothalamic, sexually differentiated nucleus (SDApc) and stereotyped vocalization exists in male Mongolian gerbils. In this present study, using unbiased stereological methods, two cytoarchitectural estimates of the SDApc's structure, neuron number, and nuclear volume were found to be sexually differentiated and also laterally asymmetrical in adult males. In ovariectomized females receiving exogenous testosterone, no cytoarchitectural component was asymmetrical. Significantly, the estimate of neuron number, but not nuclear volume, in the left SDApc of males was correlated with vocal emission rate. The authors conclude that a specific, sex-related cytoarchitectural SDApc parameter shows left-right asymmetry, suggesting the SDApc has an intimate role in mediating hemispheric specialization rather than just being an end point index of individual structural variability.

Androgenic effects on hypothalamic asymmetry in a sexually differentiated nucleus related to vocal behavior in Mongolian gerbils.

The relationship between courtship ultrasound emission rates and the volume of a discrete, sex-related, hypothalamic nucleus, the sexually dimorphic area, pars compacta (SDApc), in male and neonatally androgenized female gerbils is lateralized. Unbiased stereological estimates of neuron number and nuclear and neuropil volume are also laterally asymmetric in male SDApcs. In this study sexual differentiation and lateral asymmetry of stereologically assessed cytoarchitectural SDApc components, and their relationship to male-typical behaviors, including vocal emission, were examined in masculinized females. Female neonates received a single injection of testosterone propionate (TP) or control vehicle (Control) and were then implanted with silastic cannulae of testosterone at 65 days of age. Total SDApc volume, neuron number, nuclear volume, and neuropil volume had significantly greater values in TP compared to Control females. Neuron number was laterally asymmetric in TP females, since the left SDApc contained a greater number of smaller neurons, possibly interneurons, than the right. Courtship vocal emission and two other behaviors were masculinized in TP females. Left SDApc total volume and, most significantly, left neuron number, were correlated with vocal rates. No other lateralized correlations between behaviors and stereological estimates were found. It was concluded that various stereological parameters and the lateralization of vocal behavior and brain asymmetry depend on the early sexually differentiating effects of androgens. It is suggested that in gerbils, androgens have a role in the survival of interneurons in a laterally asymmetric hypothalamic nucleus which is an index of vocal control.

Postnatal development of a sexually dimorphic, hypothalamic nucleus in gerbils: a stereological study of neuronal number and apoptosis.

Steroid-sensitive, vocal courtship behavior is a function of a specific, hypothalamic nucleus, the sexually dimorphic area pars compacta (SDApc) in the male adult gerbil. Gender-related differences in the number of neurons in this nucleus are evident immediately after birth. By using unbiased stereological estimates of cell numbers in Nissl-stained, paraffin-wax sections of brain, we investigated the mechanisms differentiating cell number between the sexes in the SDApc on postnatal days 0, 3, 6, and 15. Cell death, identified by pyknosis, was greatest in the SDApc between days 0-3 in males, whereas in females, maximum values were reached between days 3-6. Similarly, the ratio of pyknotic to normal neurons peaked between days 0-3 in males and 3-6 in females but then declined in both sexes. Pyknotic cells were seldom seen in either sex by day 15. Morphological characteristics of apoptosis including chromatin condensation, cell fragmentation, and ingestion of apoptic bodies by macrophages were all demonstrated by transmission electron microscopy. Macrophages showed specific morphological characteristics of microglia. Cell division (mitosis) was identified in the SDApc during postnatal days 0, 3, and 6 but the numbers of mitotic figures were low, negligible on day 15, and similar between the sexes. These results demonstrate that cell death and proliferation occur simultaneously in the neonatal gerbil brain. The stereological estimates of cell death in the developing SDApc indicated a lower incidence of neuronal death occurring earlier in males than in females.

Stereological estimates of postnatal structural differentiation in a sexually dimorphic hypothalamic nucleus involved in vocal control.

Display of a specific, courtship vocalization and other masculine functions in the adult gerbil, is associated with a sexually differentiated hypothalamic nucleus, the Sexually Dimorphic Area pars compacta (SDApc). Total SDApc volume and vocal function differentiate neonatally. Since total volume is a rudimentary measure of brain nucleus differentiation, we examined the more detailed cytoarchitectural parameters behind SDApc development in gerbils, cell number, density per nucleus and individual nuclear (soma) volume. Unbiased stereological estimates were made on thick (20-40 microm) brain sections from postnatal days 1 (D1), 3 (133), 6 (DO, 16 (D16), 40 (D40) and 60 (D60) animals. Sex differences in stereological parameters were not apparent on D1 but from D3, SDApc growth patterns widely differed between the sexes. Significant differences in (i) cell number, and (ii) nuclear volume were found at D3 and D60, respectively. In males, cell number increased between D1-D6 but subsequently decreased from the D6 value by approximately 80% to reach the value of D16 which remained constant. Cell density paralleled the decrease in cell number between D6-D16 in males, whereas a progressive expansion in nuclear volume occurred between D1-D40. Male total SDApc volume enlarged between D1-D3 and D40-D60. Conversely in females, cell number and density declined between D1-D3 and D1-D40, respectively, and then remained at these low values. Cell volume, however, increased up to D40 and then significantly decreased. The resulting change to female total SDApc volume was a reduction immediately after birth, D1-D3, to a constant low value. We conclude that first, the association between various stereological measures and total SDApc volume was minimal, suggesting independent mechanisms of sexual differentiation for each cytoarchitectonic parameter. Second, the neonatal peak in SDApc cell number indicates cell migration taking place contemporaneously with cell death in males. Third, the effect of changes in cytoarchitectural components between D6-D16 and D40-D60 in males is probably due to SDApc dendritic volume expansion, suggesting that the male SDApc retains plasticity until at least puberty. Fourth, the decrease in the number of cells in females early in neonatal life, suggests programmed cell death.

Ultrasonic vocalizations in immature gerbils: emission rate and structural changes after neonatal exposure to androgen.

Emission rates of stereotyped ultrasonic vocalizations in sexually immature Mongolian gerbils are sexually dimorphic. Calling rates in interacting pairs of females are characteristically higher than in male dyads. Juvenile vocalizations produced during paired encounters show a similar spectrographic structure to adult courtship calls. Three experiments examined the developmental effects of a single injection of 100 micrograms testosterone propionate given to 0-12 h old female gerbils on total emission rates, spectrographic structure of ultrasonic vocalizations and display of other social-sexual play behaviors during encounters between immature androgenized females and stimulus partners. Reduced total rates of juvenile ultrasound emission were recorded in TP-treated females paired with stimulus males or females when compared with control male-male or female-female pairs in Experiment 1 and confirmed in Experiment 2. Male-typical play mounting was increased in pairs containing androgenized females in Experiment 2. In Experiment 3, emission rates of female-type Warble sounds were significantly decreased and the rate of male-type Steep curvilinear and Long rectilinear calls significantly increased in androgenized female and stimulus animal dyads compared with control animal pairs. New spectrographic forms of ultrasound, with readily discernible single or multiple upper harmonics, were recorded in androgenized female-stimulus partner interactions. We conclude that androgenization of female neonates (a) masculinized and also defeminized sexually dimorphic ultrasound emission rates, and virilized nonvocal behavior, suggesting a direct effect on organization of the central nervous system; (b) influenced the sonagraphic structure of calls. It is proposed that organization of juvenile vocal rates in gerbils primarily involves a number of androgen sensitive mechanisms in the central and peripheral nervous systems. However, androgenization also appeared to have an indirect role on calling rates by affecting treated female-stimulus partner relationships.

Hypothalamic distribution of astrocytes is gender-related in Mongolian gerbils.

Hypothalamic neuroglial ontogeny was examined during neonatal development of two hormone-sensitive, sex-specific nuclei, the pars compacta of the sexually dimorphic area (SDApc) and the suprachiasmatic nucleus (SCN) in the gerbil. Specific antibodies against vimentin and glial fibrillary acidic proteins (GFAP) identified neuroglia. Unbiased measures of labelled cell anatomical parameters were taken using stereomorphometric techniques. High numbers of cells in the female and male SCN immunoreacted with vimentin in neonates and GFAP in adults. Astrocytes containing vimentin or GFAP were few in number in the SDApc and surrounding areas in neonates and adults, respectively. There was a sex difference in the numerical density of both vimentin and GFAP-positive cells in the SCN. We suggest that (a) pre-astroglia are involved in gender-related organization of the SCN but not in SDApc, and (b) neuroglia have a sex-related, functional role in the mature SCN.

Lateralization of a sexually dimorphic brain area associated with steroid-sensitive behavior in the male gerbil.

Emission rates of an androgen-sensitive male courtship vocalization were positively correlated with the volume of only the left hypothalamic sexually dimorphic area, pars compacta (SDApc) nucleus in sexually active male Mongolian gerbils. The asymmetric relationship between brain nucleus and vocal behavior was confirmed in testosterone (T)-treated castrated adult males but was eliminated by castration of males and did not exist in T-treated ovariectomized adult females. Interdependence between the left SDApc volume and emission rate was specific because no significant correlations were found between (a) nonvocal precopulatory or copulatory behaviors and left or right SDApc volumes and (b) a second sexually dimorphic brain area, the suprachiasmatic nucleus and vocal or other behavioral components. The asymmetric brain-behavior relationship depends on T effects in adult males and may be lateralized by perinatal T exposure.

In vitro potency and selectivity of the non-steroidal androgen aromatase inhibitor CGS 16949A compared to steroidal inhibitors in the brain.

A sensitive in vitro 3H2O microassay for aromatase activity was used to evaluate the potency and selectivity of three aromatase inhibitors in mammalian (gerbil) and avian (ring dove) hypothalamus. The steroidal inhibitors, 1,4,6-androstatrien-3,17-dione (ATD) and 4-hydroxy-androstenedione (4-OH-A) were compared with a new non-steroidal imidazole inhibitor, CGS 16949A [4-(5,6,7,8-tetrahydroimidazo-[1,5-a]-pyridin-5-yl)benzonitrile HCl]. Adult male dove hypothalamic aromatase is highly active [Vmax = 5.3 pmol testosterone (T) converted/h/mg protein], has high substrate binding affinity (Km = 4.0 nM), and direct involvement in control of sexual behaviour. With [1 beta-3H]T or [1 beta-3H]A as substrate, male dove preoptic aromatase activity was inhibited more effectively and selectively by CGS 16949A. Thus, Kis and IC50s for aromatization were approximately 50 times lower for the non-steroidal inhibitor, and inhibition of the other major androgen-metabolizing enzymes (5 alpha/beta-reductase) occurred at concentrations at least one order of magnitude greater than for ATD and 4-OH-A. Neonatal male gerbil hypothalamic aromatase activity (Vmax = 1.3 pmol T converted/h/mg protein) was lower than in the dove. Aromatase inhibition by CGS 16949A is more potent in the neonatal gerbil than in the dove (Kis of 0.03 and 0.60 nM, respectively, with A as substrate). We conclude that the imidazole is an effective aromatase inhibitor in both the adult and developing brain.

Microimplants of estradiol in the sexually dimorphic area of the hypothalamus activate ultrasonic vocal behavior in male Mongolian gerbils.

The hormonal control of ultrasonic vocal behavior in the male Mongolian gerbil was examined by comparing the behavioral effects of androgen with those of estrogen administered to the preoptic-anterior hypothalamic area (POA-AH) in castrates. By measuring radioactivity released from solid "floating" POA-AH microimplants (mean diameter, 141 microns) of testosterone (3H-T, mean weight, 880 ng) in Experiment 1, we found that the steroid had a concentration gradient which fell rapidly from the edge of the microimplant, suggesting restricted diffusion. Using floating microimplants in Experiment 2, we studied the effects of testosterone propionate (TP, 650 ng), estradiol-17 beta benzoate (EB, 439 ng), or cholesterol (C, 478 ng) on rates of a frequency modulated ultrasonic vocalization emitted during sexual interactions. The effects on the upsweep call were compared with those on sexual mounting. The upsweep rate remained significantly below precastration levels in C implanted males. EB reinstated upsweep calling within 5 days, 3 days earlier than TP microimplants. Mounting in EB implanted males was maintained at precastration levels, whereas TP implantation restored mounting to precastration levels only after 5 days. EB was effective in inducing ultrasonic vocalizations when placed in, or near, the sexually dimorphic area (SDA) in the medial preoptic area (POM). Our results indicate that brain mechanisms underlying both ultrasonic vocalizations and mounting are directly sensitive to estradiol (E2) in the male gerbil. We conclude that E2 affects mechanisms in the SDA associated with ultrasonic calling and suggest that T is likely to act via aromatization products in the brain.

Lateralized action of androgen on development of behavior and brain sex differences.

Androgens have been linked to asymmetric brain development. Our results show that in sexually active male gerbils, the volume of the sexually dimorphic area, pars compacta (SDApc) of the preoptic region was positively correlated with the emission rate of an ultrasonic courtship vocalization. Day 1 postnatal treatment of female gerbils with testosterone propionate (TP, 100 micrograms, n = 16) or diethylstilbestrol (DES, 5 micrograms, n = 22) followed by ovariectomy and implantation of testosterone (T) filled silastic capsules in adulthood (day 120-130), increased mean volumes of the SDApc (TP, 1.21: DES, 0.84 mm3 x 10(-3)) and a second sexually dimorphic nucleus, the suprachiasmatic nucleus (SCN) (TP, 295: DES, 287.7 mm3 x 10(-3] compared to control females (n = 13) also given T in adulthood (SDApc = 0.37, SCN = 197.0 mm3 x 10(-3)). Rates of sexually dimorphic ultrasonic calls emitted during sexual interactions with estrous females (TP, 39.3: DES, 28.1 per min) were also increased relative to female controls (22.5 per min). With both treatments, significant covariances were revealed between vocalization rates and the left SDApc volumes [TP (rho), rho = .74: DES, rho = .43], but not the contralateral nucleus. Asymmetric development was absent in control females which received T as adults. Lateralization was specific to the SDApc, since no relationship was seen between either rates of calling and SCN volumes, or frequencies of nonvocal sexual components and the volumes of the SDApc or SCN. We conclude that steroid sex hormones influence lateralization of brain structure related to vocal behavior. The hormonal effect, which is likely to involve estrogen, occurs during neonatal development.

Differential effects of neonatal castration on the development of sexually dimorphic brain areas in the gerbil.

We compared the effects of neonatal castration within 6 h of birth in the Mongolian gerbil on the development of the sexually dimorphic area pars compacta (SDApc) and supraschiasmatic nucleus (SCN). Development of these brain areas was also related to a masculine courtship ultrasonic vocalization, the frequency-modulated upsweep. Castration immediately after birth resulted in differential effects with complete or partial reduction of SCN and SDApc volumes, respectively, as compared to male values. The rates of ultrasonic calling of males castrated as neonates were also decreased to female levels. In sham-operated males, calling rates were positively correlated with the volume of the left SDApc, but not the right. Both the left and the right SDApc volumes were correlated with calling rates in males castrated as neonates. The asymmetric relationship between vocal behavior and area volume was specific to the SDApc. We suggest that in the neonate (a) the sensitivity of the SDApc to the differentiating effects of androgens differs from the SCN and (b) the asymmetric link between brain structure and vocal behavior depends on the effects of androgen within 6 h of birth.

Ontogeny of sexually dimorphic ultrasonic vocalizations in Mongolian gerbils.

Sexual differentiation of emission rates (Experiment 1) and physical structure of ultrasonic vocalizations (Experiment 2) were investigated in Mongolian gerbils between 17 and 85 days of age. Animals from different litters were allowed to interact in iso- and heterosexual pairs. Vocalization rates increased in all groups, reaching a plateau at approximately day 56. Female pups had significantly higher rates of all vocalizations than male pairs before the plateau stage. Five stereotypic categories of physical structure were easily distinguished and confirmed by sound spectrographic analysis. Only mid-range frequencies of vocalizations appeared to decline throughout the juveniles' lives: this change may communicate the animal's age. Two male and one female categories were sexually dimorphic and similar in structure to those made during courtship interactions. Rates of male-type vocalizations appear to increase as testicular androgens start rising.

Development of sex differences in the response of spiny mouse pups to adult male odors.

Using a three-choice preference test, olfactory-mediated investigatory activity in response to adult male urine odor was examined in a precocially active rodent, the spiny mouse (Acomys cahirinus) aged between 3-26 days. Temporally related sex differences were seen in the time spent in the presence of the odors of father's or unfamiliar adult male's urine, or distilled (control) water. Neither male nor female pups discriminated between odors from the father and strange adult males. After the first olfactory test, when the pups were aged between four and six days, male pups strongly preferred to stay in the vicinity of urine odors of adult males, whereas female pups avoided odors of adult males and remained in the enclosure with the control odor source. To our knowledge this is the first time that such a behavioral sex difference related to olfaction has been shown to occur in young rodent pups. We suggest that the sexually dimorphic response of the pups is associated with the development of later sex differences in behavior.

Mate recognition by urine odors in the Mongolian gerbil (Meriones unguiculatus).

We examined the responses of male and female gerbils housed in opposite- or same-sex pairs to the urine odors from their cage-mate and those of an unfamiliar gerbil of the same sex. Gerbils housed in mated pairs spent more time investigating the odor of their partner. They also emitted more modulated ultrasonic vocalizations in the presence of their partner's odor than in the presence of the unfamiliar odor. Gerbils housed in same-sex pairs did not respond differentially to the odor of their cage-mate. There were sex differences in the frequency of ventral scent-marking and modulated vocalizations, with males showing more of these behaviors than females. The urine odor of the gerbil's mate can thus be discriminated from those of other gerbils. Recognition of individual odors may promote affiliative behavior and reduce aggression, thus playing an important role in pair maintenance in gerbils.

Effects of intracranial androgen on the development of masculine ultrasonic vocalizations in the Mongolian gerbil (Meriones unguiculatus).

The effects of testosterone propionate (TP) on brain mechanisms involved in the sexual differentiation of ultrasonic vocalizations were examined in Mongolian gerbils (Meriones unguiculatus). Treatment of neonatal females with TP fully masculinized the rate of emission of the upsweep precopulatory ultrasound during adult sexual interactions with oestrous females. Intracranial implantation of small crystals of TP mixed with cholesterol (65 ng) into females 1-15 h after birth also masculinized the upsweep vocalization emitted in adulthood. Implants of TP positioned in the hypothalamic area had a significantly greater masculinizing effect than TP implants outside this region, or pure cholesterol implants. Two other sexually dimorphic vocalizations, the modulated (mainly precopulatory) and unmodulated (mainly copulatory) calls were masculinized by systemic TP, but intracranial TP had no significant masculinizing action on these calls. Genital structures of females which received neonatal injections of TP were strongly virilized in that their clitorides were lengthened and male-type cornified spines were present on the glans. Females which had received intracranial implants of TP were not virilized peripherally in adulthood. We conclude that testosterone or its metabolites have a direct hypothalamic effect on the development of masculine upsweep vocalizations. Because other vocalizations were insensitive to intracranial TP, the underlying neural tissues may have different thresholds of response to androgen.

The cumulative effects of estrogen on precopulatory behavior in the female Mongolian gerbil.

Differences in responsiveness of precopulatory patterns to estradiol were investigated in the female Mongolian gerbil. Administration of five daily injections of 6 micrograms estradiol benzoate (EB) to ovariectomized females demonstrated that the precopulatory patterns differ in their responsiveness to EB. Estradiol benzoate shortened the latency to the first significant increase in frequency above post-ovariectomy levels for a group of precopulatory patterns (investigation of male's anogenital area, allogrooming and ventral gland marking) in comparison with a second group (approaching, leaving, investigation of the male's head, sand rolling, the present and piloerection postures). One component of precopulatory behavior (foot stomping) was not affected by EB. We suggest that the first group of more estrogen-sensitive patterns increases rapidly during early estrus as plasma estradiol-17 beta (E2) rises, whereas the second group shows peak levels later in estrus following the rise in plasma progesterone. We conclude that separable hormonal mechanisms may govern component precopulatory patterns.

Effects of opiate antagonists on hormones and behavior of male and female rhesus monkeys.

Opiate antagonists, naloxone (100 micrograms/kg) and naltrexone (1 mg/kg) were given to singly housed adult male or female rhesus prior to a 20-minute behavioral test with an oppositely sexed stimulus monkey. Four of the intact adult males were socially and sexually experienced. The remaining two intact males and two castrated males had been reared in socially restricted conditions and were psychosexually deficient. Adult females were ovariectomized, and the effects of opiate antagonists were examined with or without concurrent estradiol treatment. Both antagonists inhibited sexual behavior of the socially reared, sexually active, intact males. No stimulatory effects on sexual behavior were observed for sexually deficient males, whether intact or castrated. Females showed little change in sexual behavior following opiate antagonist treatment, regardless of endocrine status. The proportion of approaches of the female to the male was increased when naloxone, but not naltrexone, was given. Specific endocrine effects of the opiate antagonists were only found in intact males. Naltrexone significantly increased LH concentrations in the two males tested, while the increase in LH in the four males receiving naloxone was not significant. In all intact males, increases in LH were accompanied by statistically significant increases in circulating concentrations of testosterone following naloxone and naltrexone. The gonadotropic stimulating effect of the opiate antagonists was specific to LH, and no changes were observed in circulating concentrations of FSH in either sex.

The regulation of precopulatory behavior by ovarian hormones in the female Mongolian gerbil.

The hormonal regulation of precopulatory behavior in the female Mongolian gerbil was studied using two groups (N = 6) of sexually experienced females. A novel testing procedure was used which involved females living continuously with test males for several days. The test males showed either full sexual behavior (copulating males, C) or only precopulatory behavior (noncopulating males, NC). Experiment 1 investigated changes during the estrous cycle and following ovariectomy in females. Experiment 2 studied the effects of hormonal treatment of these ovariectomized females with 6 micrograms estradiol benzoate (EB) followed by 0.4 mg progesterone (P) or by 0.04 ml arachis oil. When tested with NC males, females displayed a greater range of precopulatory behavior. The patterns could be classified into three groups according to the manner of response to ovariectomy and hormone treatment. Group I patterns (approach, leave, and olfactory investigation of the male's head) were affected by neither ovariectomy nor EB treatment relative to Day 3 levels (Day 3, day preceding estrus; Day 4, estrus), but they were increased to estrous levels by EB and P. Group II patterns (darting, foot-stomping, and the present and piloerection postures) appeared only during estrus, did not appear after ovariectomy, and reappeared only after sequential EB and P treatment. Group III patterns (investigation of the male's anogenital area, allogrooming, ventral gland marking, and sand-rolling) were reduced relative to both estrus and Day 3 levels by ovariectomy and increased above Day 3 levels by EB alone; EB and P treatment further increased Group III patterns to the level of estrus. It is suggested that female precopulatory behavior patterns differ in their responsiveness to ovarian hormones. Estrogen appears to affect those patterns associated with the earliest stages of estrus (Group III).