Estradiol and testosterone inhibit rat seminiferous tubule development in a hormone-specific way

Follicle stimulating hormone (FSH), testosterone (T) and estradiol (E₂) are known to regulate testis maturation, and changes in FSH secretion induced by sex steroid treatment may mediate the effects of sex hormones. The aim of this study was to compare the effects of T and E₂ on the pre-meiotic steps of first spermatogenesis and FSH level in rats. Male rat pups were injected daily with 17β-estradiol benzoate (EB; 12.5 μg) or testosterone propionate (TP; 2.5 mg) with the use of one of the two administration modes: 1/transient mode; hormone injections on postnatal days (PND) 1–5 followed by daily vehicle injections until PND 15 (t-EB and t-TP, respectively) or 2/continuous mode; hormone injections on PND 1–15 (c-EB and c-TP, respectively). The control group was injected with vehicle alone. On PND 16, blood was taken for serum hormone measurement and testes were collected for analysis of seminiferous tubule morphometry as well as cell number, proliferation and apoptosis. Testis weight, tubule length, Sertoli and germ cell numbers were reduced, and cell apoptosis in seminiferous epithelium was increased after transient EB and TP treatments. Despite normal or increased FSH secretion, the c-EB treatment inhibited pre-meiotic germ cell development and augmented cell apoptosis, whereas the c-TP treatment reduced the spermatocyte number and inhibited the formation of seminiferous tubule lumen. In conclusion, transient administration of EB or TP during PND 1–5 inhibited testis growth, whereas continuous administration (PND 1–15) impaired pre-meiotic germ cell development in a hormone-specific way.     

Cytological and expression studies and quantitative analysis of the temporal and stage-specific effects of follicle-stimulating hormone and testosterone during cocultures of the normal human seminiferous epithelium

In vitro culturing of normal human seminiferous epithelium remains largely unexplored. To study normal human spermatogenesis in vitro, we used a micromethod for the purification and culture of Sertoli cells, spermatogonia A, spermatocytes, and early round spermatids. Cytological quantitative data for Sertoli and premeiotic germ cell cocultures isolated from normal testicular biopsies demonstrated that cells were able to proliferate (4%), complete meiosis (6.7%), and differentiate into late round (54%), elongating (49%), and elongated (17%) spermatids at similar in vivo time delays (up to 16 days) in response to FSH + testosterone stimulation. Cells maintained normal meiotic segregation, chromosome complements, and specific gene expression profiles. Follicle-stimulating hormone + testosterone stimulated spermatogonia proliferation and Sertoli cell survival. Follicle-stimulating hormone and especially FSH + testosterone increased diploid germ cell survival during the first week, whereas only FSH + testosterone was able to inhibit cell death during the second week of culture. Follicle-stimulating hormone and especially FSH + testosterone also stimulated meiosis resumption, although this was restricted to late pachytene and secondary spermatocytes. In contrast, spermiogenesis was only stimulated by FSH + testosterone. Expression studies showed that apoptosis was induced in the nucleus of diploid cells, and in nuclear and cytoplasmic compartments of spermatids, mainly triggered by the Fas pathway. Although junctional complexes between Sertoli and premeiotic germ cells were partially reacquired, the same did not apply to spermatids, suggesting that FSH potentiated by testosterone was unable to render Sertoli cells competent to bind round spermatids.     

The effects of testosterone propionate and estradiol benzoate on the vitro synthesis of FSH.

The effects of estradiol benzoate (EB) and testosterone propionate (TP) on pituitary and plasma concentrations of follicle stimulating hormone (FSH) and the in vitro synthesis of FSH in pituitary tissue was studied in mature male rats. By the 4th day of treatment with EB, pituitary content and concentration of FSH had declined, and content had fallen to 6% and concentration to 3% of pretreatment values. Similar results occurred during in vitro synthesis. However, serum levels of FSH did not show any decline until the 21st and 28th days of treatment. Administration of TP produced a progressive increase in pituitary content and concentration of FSH, though serum levels remained unchanged for the 1st 7 days, after which they fell slightly. The effect of TP on the in vitro synthesis of FSH showed no consistent pattern, though in no case was a decrease in the uptake of labeled leucine into immunoprecipitable FSH observed. The results suggest that EB and TP have different effects on pituitary FSH in normal adult male rats.     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone‐filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 502–510, 2003     

Organizational effects of testosterone,estradiol, and dihydrotestosterone on vasopressin mRNA expression in the bed nucleus of the stria terminalis

In adulthood, male rats express higher levels of arginine vasopressin (AVP) mRNA in the bed nucleus of the stria terminalis (BST) than do female rats. We tested whether this sex difference is primarily due to differences in neonatal levels of testosterone. Male and female rats were gonadectomized on the day of birth and treated with testosterone propionate (TP) or vehicle on postnatal days 1, 3, and 5 (P1, P3, and P5). Three months later, all rats were implanted with testosterone-filled capsules. Two weeks later, brains were processed for in situ hybridization to detect AVP mRNA. We found that neonatal TP treatment significantly increased the number of vasopressinergic cells in the BST over control injections. We then sought to determine the effects of testosterone metabolites, estradiol and dihydrotestosterone, given alone or in combination, on AVP expression in the BST. Rat pups were treated as described above, except that instead of testosterone, estradiol benzoate (EB), dihydrotestosterone propionate (DHTP), a combination of EB and DHTP (EB+DHTP), or vehicle was injected neonatally. Neonatal treatment with either EB or EB+DHTP increased the number of vasopressinergic cells in the BST over that of DHTP or oil treatment. However, treatment with DHTP also significantly increased the number of vasopressinergic cells over that of oil treatment. Hence, in addition to bolstering evidence that estradiol is the more potent metabolite of testosterone in causing sexual differentiation of the brain, these data provide the first example of a masculinizing effect of a nonaromatizable androgen on a sexually dimorphic neuropeptide system.     

Hormonal regulation of spermatogenesis and spermiogenesis

Normal testicular function is dependent upon hormones acting through endocrine and paracrine pathways both in vivo and in vitro. Sertoli cells provide factors necessary for the successful progression of spermatogonia into spermatozoa. Sertoli cells have receptors for follicle stimulating hormone (FSH) and testosterone which are the main hormonal regulators of spermatogenesis. Hormones such as testosterone, FSH and luteinizing hormone (LH) are known to influence the germ cell fate. Their removal induces germ cell apoptosis. Proteins of the Bcl-2 family provide one signaling pathway which appears to be essential for male germ cell homeostasis. In addition to paracrine signals, germ cells also depend upon signals derived from Sertoli by direct membrane contact. Somatostatin is a regulatory peptide playing a role in the regulation of the proliferation of the male gametes. Activin A, follistatin and FSH play a role in germ cell maturation during the period when gonocytes resume mitosis to form the spermatogonial stem cells and differentiating germ cell populations. In vitro cultures systems have provided evidence that spermatogonia in advance stage of differentiation have specific regulatory mechanisms that control their fate. This review article provides an overview of the literature concerning the hormonal pathways regulating spermatogenesis.     

Gonadal hormones stimulate ultrasound production by female hamsters.

Two studies were conducted to examine the role of sex hormones in ultrasound production by female hamsters. Ovariectomized hamsters were treated with estradiol benzoate (EB), testosterone propionate (TP), progesterone (P), estradiol plus progesterone (EB + P), testosterone plus progesterone (TP+P), or oil vehicle. Rates of ultrasound production by these females were observed in response to brief male-female contact, and during exposure to synthetic ultrasounds. Maximal rates of ultrasound production required EB + P or TP+P replacement therapy. Intermediate call rates were stimulated by EB and TP individually, whereas P alone had no significant effect. These results support the hypothesis that ovarian estrogens and progesterone control proceptive as well as receptive behaviors in female hamsters.     

Follicle-stimulating hormone regulates both Sertoli cell and spermatogonial populations in the adult photoinhibited Djungarian hamster testis

The hormones that regulate spermatogonial development are ill defined, in part due to lack of appropriate experimental models. The photoinhibited hamster model provides a rich source of spermatogonia, thus making it an ideal model to study their control. This study aimed to assess the effects of FSH, in the absence of testosterone, on the reinitiation of Sertoli cell and spermatogonial development in the photosensitive adult Djungarian hamster. Hamsters raised under long photoperiods (LD, 16L:8D) were exposed to short photoperiods (SD, 8L:16D) for 11 wk, leading to suppression of gonadotropins and regression of testicular function. Groups of 10 animals then received FSH alone or in combination with the antiandrogen, flutamide, for 7 days. Two control groups maintained either under long or short photoperiods were treated with vehicle. Sertoli and germ cell number were then determined using the optical disector (sic) stereological technique. The number of Sertoli cells, type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes were suppressed in SD controls to 66%, 34%, 19%, and 10% (all P < 0.01) of long-day control values, respectively. Later germ cell types were not detected. FSH treatment, with or without flutamide, increased Sertoli cell number (P < 0.01) to normal long-day values. Similarly, FSH treatment in the absence/presence of flutamide increased type A spermatogonia, type B spermatogonia/preleptotene spermatocytes, and leptotene/zygotene spermatocytes to approximately 85%, 69%, and 80% (all P < 0.01) of long-day controls, respectively. Our data demonstrate that the reinitiation of spermatogonial maturation in this model is dependent on FSH in the presence of an antiandrogen. Surprisingly, the adult Sertoli cell population in this model is also hormone dependent. This naturally occurring model provides a unique opportunity to understand the mechanisms (apoptotic and/or proliferative) by which FSH regulates Sertoli and germ cell development in the adult animal.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Prolactin modulates steroidogenesis by rat granulosa cells: II. Effects on estradiol

The effects of prolactin on secretion of estradiol by rat granulosa cells obtained at two stages of differentiation and cultured under various conditions were determined. Relatively undifferentiated granulosa cells were obtained from immature, diethylstilbestrol-treated, hypophysectomized (HPX) rats and cultured in serum-free medium or with 10% serum. More highly differentiated granulosa cells were obtained on the morning of proestrous from the preovulatory follicles of immature rats in which an estrous cycle had been induced with 4 IU pregnant mare's serum gonadotropin; these cells were cultured in medium containing 10% serum. Cells were cultured for 3 days with graded doses of prolactin (0, 0.02, 0.2, 2, or 10 micrograms/ml) alone or in combination with follicle-stimulating hormone (FSH; 300 ng/ml), testosterone (0.5 microM), or FSH + testosterone. In control cultures (no prolactin) the relatively undifferentiated granulosa cells from HPX rats secreted negligible quantities of estradiol except when both FSH and testosterone were supplied. Prolactin alone or in combination with FSH or testosterone had no effect on estradiol secretion, but prolactin in combination with FSH + testosterone significantly decreased secretion in a dose-dependent fashion. This set of prolactin treatments was applied in both serum-free medium and medium containing 10% serum, with similar results under both culture conditions. The inhibitory effects of prolactin appeared to be reversible if cells were cultured with prolactin for only 1 day, but were not reversed if cells were cultured with prolactin for 2 or 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential response of testis and serum gonadotropins to testosterone in rats treated with a gonadotropin releasing hormone antagonist or estradiol 17 beta.

Adult rats treated with a GnRH antagonist (Ac D2Nal1, D4Cl Phe2, DTrp3, DArg6, DAla10 GnRH; code: 103-289-10, National Institutes of Health, USA) for 5 weeks (250 micrograms/kg b.w) showed multiple degrees of impairment and atrophy of the genital organs concomitant with decreased serum levels of testosterone, LH and FSH. Inhibition of spermatogenesis was characterized by germ cell degeneration and overall decline in different cell numbers and in particular, spermatids of any kind were completely absent. Testosterone supplementation (60 micrograms/rat/day, sc) to GnRH antagonist treated rats, for the same period, significantly elevated the weights of the sex organs, and the serum levels of hormones. Spermatogenesis was improved both qualitatively and quantitatively; albeit failed to be restored back to control levels. Treatment with estradiol 17 beta (1 microgram/rat/day) for 5 weeks had insignificant effect on spermatogenesis but the weights of the genital organs (seminal vesicles by 19% and ventral prostate by 40%) and the levels of serum hormones (LH by 24%, FSH 22% and testosterone by 25%) were otherwise reduced. Administration of testosterone either alone or in combination with estradiol 17 beta had only a marginal effect on spermatogenesis or on other reproductive parameters. The results indicate a positive shift in the response of the testis and serum levels of gonadotropins to testosterone supplementation in rats treated with either GnRH antagonist or estradiol 17 beta.     

Mounting and receptive behavior in the ovariectomized female rat: Influence of estradiol,dihydrotestosterone, and genital anesthetization

Two experiments were performed with ovariectomized female rats in an attempt to determine whether estradiol and dihydrotestosterone work synergistically in the brain to activate mounting behavior. In Expt 1, performed in Göteborg, it was found that females treated daily with 2 μg estradiol benzoate (EB) combined with 500 μg dihydrotestosterone (DHT) displayed significantly more mounts with pelvic thrusting than other females treated with the oil vehicle, 500 μg DHT, or 2 μg EB. The behavior of rats receiving EB + DHT was indistinguishable from that of yet another group of females which received 200 μg testosterone propionate (TP). In Expt 2, performed in Rotterdam, it was found that ovariectomized female rats treated with either 200 μg TP or 2 μg EB + 200 μg dihydrotestosterone propionate (DHTP) mounted significantly more than females treated with 2 μg EB. Both clitoral size and the growth of cornified papillae on the glans clitoris were stimulated by the administration of TP or EB + DHTP. However, in no group was the frequency of mounting affected by anesthetization of the clitoris and external vagina with lidocaine paste. Lordosis quotients of females treated with EB + DHTP were significantly lower than in rats receiving either EB or TP, again regardless of whether or not the genital region was anesthetized. It is concluded that the effects of DHT on estradiol-induced mounting and receptivity most likely result from the action of this androgen on the brain, and not from the stimulatory effect which DHT may exert on genital sensory receptors.     

Blockade of lordosis by androst-1,4,6-triene-3,17-dione (ATD) and tamoxifen in female hamsters primed with testosterone propionate

Normal female hamsters display lordosis after testosterone propionate (TP) plus progesterone (P) treatments. Such effect is probably mediated through aromatization of testosterone (T) into estradiol. If so, then an aromatase inhibitor (ATD) or an estrogen antagonist (tamoxifen, TAM) should be able to block the activational effect of T on lordosis. To test this hypothesis, 48 ovariectomized female hamsters were assigned into six groups which, according to treatments received, were ATD + TP, TAM + TP, OIL + TP, ATD + EB (estradiol benzoate), TAM + EB, and OIL + EB groups. The groups received assigned treatments for 2 days and were injected with P on the third day. Five minutes of behavior test was conducted 4 hr after P injection. The OIL + TP, OIL + EB, and ATD + EB groups all had averaged total lordosis duration (TLD) longer than 200 sec. The TLD of the TAM + EB group was only 117 sec. The ATD + TP and TAM + TP groups showed almost no lordosis. The results showed that the estrogen antagonist (TAM) impaired lordosis no matter whether the animals were primed with TP or EB, but the aromatase inhibitor (ATD) blocked lordosis only in TP primed females. It is concluded that the aromatization of T to estrogen is required for testosterone activation of lordosis in female hamsters.     

Enhanced ERbeta immunoexpression and apoptosis in the germ cells of cimetidine-treated rats

Background  

Cimetidine, refereed as antiandrogenic drug, causes hormonal changes in male patients such as increased testosterone and FSH levels. In the rat testis, structural alterations in the seminiferous tubules have been related to germ cell loss and Sertoli cell death by apoptosis. Regarding the important role of Sertoli cells in the conversion of testosterone into estrogen, via aromatase, the immunoexpression of estrogen receptors-beta (ERbeta) was evaluated in the germ cells of untreated and treated rats with cimetidine. A relationship between ERbeta immunoreactivity and apoptosis was also investigated in the germ cells of damaged tubules.     

Role of FSH and triiodothyronine in Sertoli cell development expressed by formation of connexin 43-based gap junctions

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Connexin 43 (Cx43) is the most ubiquitous constitutive protein of gap junctions in the testis. This study evaluates the effects of the hyperstimulation of FSH and T3 during testicular maturation on Cx43 expression in the testis. The newborn, male Wistar rats were divided randomly into four experimental groups: FSH group-daily injections of FSH 7.5?IU/animal; T3 group-100?μg T3/kg body weight; FSH+T3 group-both substances; A control group-received vehicles in the same volume. Proliferating cell nuclear antigen immunohistochemistry and toluidine blue staining were used to determine the germ cell proliferation and degeneration. Cx43 immunolocalization was evaluated to find Cx43 maturational changes. Under FSH treatment, the proliferation rate was high so the total number of Sertoli cells increased with a low level of degeneration and lumen formation. T3 stimulation evoked a reduction in the proliferation rate and a decrease in Sertoli cell number but with intensive formation of lumen. T3+FSH inhibited the proliferation rate and stimulated lumen formation together with degeneration, which negatively influenced the number of germ cells in the seminiferous epithelium. We conclude that T3 action seems to be particularly connected with the maturation of Cx43 gap junctions. FSH stimulates maturation of Sertoli cell function, but this effect may take place regardless of the presence of Cx43-dependent intercellular communication. The hyperstimulation of both FSH and T3 damages Cx43 connections and hence evokes regressional changes in the seminiferous epithelium.     

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.     

Postcastration rise in plasma gonadotropins is blocked by a luteinizing hormone-releasing hormone antagonist

The dependence of the acute increases in plasma gonadotropins following castration on luteinizing hormone-releasing hormone (LHRH) was assessed with the use of a potent LHRH antagonist [ALHRH; (Nac-L-Ala1,p-Cl-D-Phe2,D-Trp3,6) LHRH]. Blood samples were collected from male and female rats at the time of castration and 2, 4, 8, 12, 24 and 48 h following and plasma gonadotropin levels were determined. Immediately following castration (diestrus I for females) animals received one of the following treatments: females-vehicle, 100 micrograms ALHRH, 50 micrograms estrogen benzoate (EB), or 100 micrograms ALHRH + 50 micrograms EB; males-vehicle, 100 micrograms ALHRH, 500 micrograms testosterone propionate (TP), or 100 micrograms ALHRH + 500 micrograms TP. ALHRH blocked the selective increase in plasma follicle-stimulating hormone (FSH) observed in female rats as well as the parallel increases in both gonadotropins seen in male rats following castration. Administration of EB or ALHRH + EB to females significantly suppressed both gonadotropins compared with control levels. However, EB alone did not completely block the rise in plasma FSH in females. In males, all three treatments significantly suppressed the increases in both gonadotropins when compared with control levels. These data demonstrate that hypothalamic LHRH plays an essential role in the acute elevations of plasma gonadotropins following castration in rats. In addition, these data suggest that the selective rise of FSH in females is dependent on LHRH stimulation of pituitary gonadotropes.     

Activation of sexual behavior by implantation of testosterone propionate and estradiol benzoate into the preoptic area of the male Japanese quail (Coturnix japonica)

Intracranial implantation of minute pellets of gonadal steroids was performed to determine neuroanatomical loci at which steroids activate sexual behavior in the Japanese quail (Coturnix japonica). In this species, systemic treatment of castrated males with either testosterone propionate (TP) or estradiol benzoate (EB) restores male-typical copulatory behavior (head grabbing, mounting, and cloacal contact movements). In addition, EB activates female-typical receptive behavior (crouching). Adult male castrated quail were implanted intracranially with 300-micrograms pellets containing TP, EB, or cholesterol (CHOL) and behavior was tested with intact males and females. Either TP or EB pellets in the preoptic area (POA) activated male-typical copulatory behavior. Mounting was specifically activated without concomitant activation of other steroid-sensitive sexual and courtship behaviors. TP and EB implants in adjacent nuclei containing receptors for these steroids and CHOL implants in POA had no effect on male-typical copulatory behavior. Eighteen percent of all males tested for female-typical receptivity crouched, but no specific effect of EB was seen at any site. The similarity of the POA sites for activation of mounting by TP and EB is consistent with the hypothesis that cells within the POA aromatize testosterone to estrogens, which directly stimulate the cellular processes leading to behavioral activation.     

Effects of estradiol benzoate in combination with dihydrotestosterone on postejaculatory vocalization and refractory period in castrated male rats

The objective of this experiment was to compare the effects of estradiol ben-zoate (EB) treatment with those of testosterone propionate (TP) on the postejaculatory vocalization and refractory period in castrated dihydrotestosterone (DHT)-treated male rats. Twelve reliable maters were tested, castrated, and then treated with subcutaneous implants of DHT and daily injections of either 200 μg of TP (N = 6) or 200 μg of EB (N = 6). Testing continued weekly for 9 weeks with treatments interchanged after the fourth week. EB treatment resulted in: (1) a reduction in the number of males that vocalized, (2) a reduction in the duration of vocalization, and (3) the exhibition of extraordinarily abbreviated postejaculatory refractory periods by a few males. It was suggested that high doses of estradiol act to counter inhibitory processes during the refractory period.