Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.     

Follicular development and a single ovulation induced with pulsatile administration of Gn-RH in anovulatory women: studies of hormonal analysis and follicular sonometry

The method of pulsatile administration of gonadotropin-releasing hormone (Gn-RH) has been proven as a useful means for induction of ovulation in anovulatory women. In our series of clinical trials, 23 out of 29 anovulatory patients ovulated with pulsatile administration of Gn-RH. Seven patients who ovulated volunteered for the present study with daily hormonal analysis and follicular sonometory . Two patients have oligomenorrhea, 3 patients secondary amenorrhea-1st grade (the sole administration of gestagen required for withdrawal bleeding) and the remaining 2 patients secondary amenorrhea-2nd grade (the combined administration of estrogen and gestagen required for withdrawal bleeding). A diagnosis of hyperprolactinemia was made for one patient with secondary amenorrhea-1st grade. Pulsatile administration of Gn-RH was performed by the use of a self-administered infuser . The infuser was connected to an i.v. indwelling catheter via a specially designed blood backflow eliminater . Five micrograms or less of Gn-RH was given every 2 hr from 07:00 to 23:00 hr daily. Five patients received HCG during the preovulatory period. In one patient, a short term treatment of HMG was added to Gn-RH treatment. Follicular sonometry revealed the development of a single dominant follicle which reached between 20 and 28 mm (23.7 +/- 0.12 mm, mean +/- S.E.) in diameter at the preovulatory period. Disappearance of a dominant follicle was recognized in the early luteal phase. Characteristic increases in estradiol were recognized concomitantly with the development of a dominant follicle. Progesterone levels after ovulation were within the limits of its normal "luteal phase" rise. The present data suggest that pulsatile administration of low dose Gn-RH with nocturnal interruption of treatment is effective for normal progress of follicular development in various types of anovulatory patients, culminating in single ovulation. This paper includes the discussion on our method which may be responsible for a high success rate of ovulation induction.     

Aromatase inhibitors in ovarian stimulation

The selective estrogen receptor modulator, clomiphene citrate (CC), has been the principal drug used for induction of ovulation in women with polycystic ovarian syndrome (PCOS). CC is associated with adverse side effects and low pregnancy rates attributed to long-lasting estrogen receptor depletion. Anastrozole and letrozole are potent, non-steroidal, reversible aromatase inhibitors, developed for postmenopausal breast cancer therapy. We hypothesized that aromatase inhibitors could mimic the action of CC in reducing estrogen negative feedback on follicle stimulating hormone (FSH) secretion, without depleting estrogen receptors. In a series of preliminary studies, we reported the success of aromatase inhibition in inducing ovulation in anovulatory women with PCOS. Moreover, we showed that concomitant use of aromatase inhibitors resulted in a significant reduction of the FSH dose needed for controlled ovarian hyperstimulation. We suggest that aromatase inhibitors act through an increase in endogenous gonadotropin secretion as well as through increased intraovarian androgen levels that may increase ovarian FSH receptors. Recently, we demonstrated the safety of aromatase inhibitors in pregnancy outcome studies examining spontaneous pregnancy loss, multiple pregnancy rates and congenital anomalies compared to a control group of infertility patients treated with CC.     

Inhibition of aromatase: insights from recent studies

Aromatase is the rate limiting enzyme that catalyzes the conversion of androgens to estrogens. Blockade of this step allows treatment of diseases that are dependent upon estrogen. Over the past two decades, highly potent and specific aromatase inhibitors have been developed which block total body aromatization by over 99%. An important recent question is whether aromatase inhibitors are superior to the antiestrogens for treatment of hormone-dependent breast cancer. The third generation aromatase inhibitors have been compared to tamoxifen for the treatment of breast cancer in the advanced, adjuvant, and neoadjuvant settings. All of these studies suggest the superiority of aromatase inhibitors over tamoxifen. The mechanism responsible for the superiority of the aromatase inhibitors relates to the estrogen agonistic effects of tamoxifen. During exposure to estrogen deprived conditions and to tamoxifen, breast cancer cells adapt and upregulate the MAP kinase and PI-3 kinase pathways. These growth factor signaling pathways potentiate the estrogen agonistic properties of tamoxifen. Data from a large adjuvant therapy trial (ATAC trial) provide evidence that the aromatase inhibitors may also be superior for breast cancer prevention. The mechanism for superiority in this setting probably relates to the genotoxic effects of estradiol metabolites. The aromatase inhibitors may be also useful for the treatment of endometriosis and for ovulation induction as evidenced by preliminary data. The recent advances in development of the aromatase inhibitors clearly demonstrate the utility of these agents for treatment of breast cancer and potentially for other indications.     

Steroids and follicular rupture at ovulation

The preovulatory surge of gonadotropins stimulates follicular steroidogenesis and changes from estrogen as the major product to progesterone. We shall overview the studies dealing with the role of ovarian steroidogenesis in follicular rupture at ovulation. Several inhibitors of steroidogenesis blocked follicular rupture in vivo. Likewise, RU 38486 partially blocked ovulation triggered by hCG. Collectively, these data support the knowledge that follicular steroidogenesis is required for ovulation. Recent studies confirmed the essential role of plasminogen activator (PA) in follicular rupture. The LH stimulation of PA activity was partially blocked by several inhibitors of steroidogenesis and it could be restored by the addition of progesterone, testosterone and estradiol-17 beta, but not the non-aromatizable 5 alpha-dihydrotestosterone. Gonadotropic stimulation enhanced only the synthesis of tissue type PA (t-PA) and not that of urokinase. Likewise, inhibition of steroidogenesis, reduced only the synthesis of t-PA and was reversed by addition of estradiol-17 beta. It seems, therefore, that follicular steroids, most probably estrogen, are involved in the preovulatory rise in follicular t-PA activity.     

Ovulation in light-estrous rats induced by darkness

Adult female rats show continual vaginal cornification and cease ovulation a few weeks after they are exposed to continuous lighting (light-estrous rats). When these rats were placed in the darkness for 10 hr, 80% of the animal ovulated approximately 46 hr later. Peripheral LH increased to a small peak immediately after placing in darkness concomitant with a decrease in pituitary LH content; a large peak, 20 times higher than the basal LH level, was observed at 20 to 22 hr. Progesterone concentration in ovarian vein blood remained at extremely low levels while estrogen levels tended to rise after small LH peak. This estrogen rise appeared to play an important role in inducing the main LH peak. Simulation of the small LH peak by low doses of exogenous LH succeeded in inducing ovulation of light-estrous rats in similar fashion to the exposure of light-estrous rats to 10-hr darkness. Therefore, the small amount of LH secretion observed after the initiation of the darkness-treatment may be considered as a trigger for the whole sequence of hormonal changes leading to ovulation.     

Hormonal contraception

The combination hormonal contraceptive pill consisting of a fixed ratio of a progestational compound to an estrogenic compound administered from the 5th to 25th day of the cycle approaches 100% in efficacy. Cycle control is excellent and is related to the mechanism of action. The progestogen plus estrogen of the combination pill inhibit the hypothalamic-anterior pituitary-ovarian axis, thus insuring almost complete inhibition of ovulation and endogenous steroid hormone biosynthesis. The lack of endogeneous hormone is not critical since the pill replaces the lost hormones particularly as to uterine endometrial stimulation so that the tissue is prepared for a bleed on treatment withdrawal. The carefully timed hormonal replacement ensures excellent cyclicity. Although suppression of ovulation may be continued for years, cessation of treatment is followed by a return to normal hormonal function and fertility.Variants of the combination treatment include a once-a-month pill and a once-a-month injectable preparation. Both formulations are based on combinations of a progestogen plus estrogen.A high degree of efficacy approaching that seen for the combination pill has been achieved with a sequential regimen. In this procedure an estrogen alone is administered for 5 to 15 days while a mixture of estrogen plus a progestational agent is administered for the balance of the 20 days of treatment. Withdrawal of treatment brings on a bleed in 2 to 5 days. The mechanism of of antifertility is similar to that of the combination pill.Contraceptive efficiency of a high order may be attained with a small continuous dose of a progestational agent. The pregnancy rate and cyclicity are acceptable but not as good as that of the combination or sequential regimens. The pure progestogen treatment has been adapted to a pseudo post-coital therapy where one sexual contact requires one pill usually within 1 to 3 hours of the event. Thus far a reasonable efficacy has been established but as much as 33% of the cycles may be less than 20 days in length.A Single injection of a progestogen can produce effective antifertility for 90 days. This treatment is efficacious but the early treatment periods may involve considerable irregular bleeding and after repeated use ammenorrhea may be a problem.A true post-coital treatment has not been established. However, for emergency use 4 to 6 days of treatment with high doses of estrogen is highly effective in preventing pregnancy. The mechanism may involve speeding of ova transport thus preventing implantation.     

Induction of ovulation and spermatogenesis by hMG/hCG in hypogonadotropic GH-deficient patients.

Nine female and 20 male hypogonadotropic GH-deficient patients were studied for sexual development by hCG/hMG. In the female patients, gonadotropin therapy was started at the mean age of 22.7 +/- 2.1 years. The administration of progesterone induced withdrawal bleeding at an average of 2.77 +/- 1.94 years after the initiation of hMG/hCG therapy in 8 of the 9 patients studied. Of 6 patients who had been confirmed as positive in a gestagen test, induction of ovulation by hMG/hCG was observed in 5 patients at an average of 5.58 +/- 1.23 years after the onset of therapy, but not in the remaining patient who had been given estrogen and progesterone 4 years 9 months prior to the initiation of the gonadotropin therapy. In male patients, gonadotropin therapy was started at the mean age of 23.6 +/- 5.7 years. Seminal fluid was obtained by masturbation and brought to our clinic in the morning. Of the 20 patients, 19 patients could be observed once a month regularly. Of the 19 patients, spermatozoa could be detected at a mean period of 2.19 +/- 0.87 years after initiation of hCG/hMG therapy in 18, but not in the remaining patient, after 5 years of therapy, who did not receive hCG/hMG regularly. The sperm count exceeded 20 x 10(6)/ml and more in 12 and was lower than that in 8 patients after 3 years of the therapy. No side effects were observed in female patients, but gynecomastia developed in 2 of the 20 male patients. These data suggest that gonadotropin therapy for hypogonadotropic GH-deficient patients is effective in promoting ovulation and spermatogenesis despite the initial replacement therapy with sex hormones.     

Effects of ventromedial nucleus lesions on estradiol induced ovulation in the rat

Small bilateral stereotaxic lesions were made in the hypothalamic ventromedial nucleus (SVMN) to determine: (i) whether estrogen would restore early receptivity in unreceptive SVMN lesioned female rats and (ii) whether SVMN lesions would suppress estrogen induced ovulation in the rat. SVMN lesions were shown to completely suppress spontaneous early receptivity and seriously impair estrous receptivity in 5-day cyclic female Wistar rats. A loss of early receptivity in response to 10 μg estradiol benzoate (EB) was also observed in SVMN lesioned females, in comparison to unoperated, sham VMN and dorsomedial nucleus (DMN) lesioned animals. Isolated SVMN lesioned females exhibited a weak ovulatory response to 10 μg EB, but, where shown to be unreceptive prior to estrogen injection, they never ovulated. On the contrary, ovulation occured in about 50% of cases in isolated unoperated and in sham VMN and DMN lesioned females following estrogen administration. The mechanisms whereby EB brought about precocious ovulation in 5-day cyclic female rats were therefore concluded to be dependent on VMN functional integrity and thereby on the degree of early sexual receptivity in the rat.     

The Unique Impact of Menstruation on the Female Voice: Implications for the Evolution of Menstrual Cycle Cues

Research shows that hormonal changes in women across the menstrual cycle affect vocal production. Most work has documented shifts at high fertility times (i.e., ovulation) or during premenstruation. However, hormonal changes at menstruation also affect female physiology and behavior and could affect vocal production. The present studies investigated perceptual differences in voices recorded during menstruation compared with recordings taken at other times of the menstrual cycle. Results show that male raters could reliably identify voices recorded during menstruation with or without the presence of a voice recorded closest to ovulation. In addition, voices recorded at menstruation were identified as being the most unattractive. These findings indicate that voice recordings taken at times of lowest fertility may uniquely impact women’s vocal production and that perceptions of voice based on cycle phase are not specific to the time of heightened fertility. Implications for the evolution of human menstrual cycle cues are discussed.     

Attunement to the fertility status of same-sex rivals: women's testosterone responses to olfactory ovulation cues

Evolutionary theories of mating suggest that changes in fertility across the menstrual cycle play an important role in sexual selection. In line with this framework, the current research examined whether olfactory cues to the fertility of a same-sex rival would prompt hormonal signs of intrasexual competition in women. Women exposed to the scent of another woman close to ovulation subsequently displayed higher levels of testosterone than women exposed to the scent of a woman far from ovulation. Whereas women exposed to the scent of a woman in the mid-luteal phase displayed sizable decreases in testosterone over time, no such decline was observed among women exposed to the scent of a woman near ovulation. Thus, olfactory cues signaling a rival's heightened level of fertility were associated with endocrinological responses in women that could be linked to intrasexual competition.     

Nestorone: clinical applications for contraception and HRT

The 19-nor derivatives of progesterone are referred to as "pure" progestational molecules as they bind almost exclusively to the progesterone receptor (PR) without interfering with receptors of other steroids. In this category is Nestorone, which has strong progestational activity and antiovulatory potency with no androgenic or estrogenic activity in vivo. These properties make it highly suitable for use in contraception and hormonal therapy (HT). Due to its high potency, very low doses of Nestorone may be delivered via long-term sustained-release delivery systems. Nestorone, 75 or 100 microg per day, released by vaginal ring has suppressed ovulation in women, with inhibition of follicular maturation. A vaginal ring releasing both 150 microg of Nestorone and 15 microg of ethinyl estradiol per day has effectively suppressed ovulation for 13 consecutive cycles. Nestorone has also been used effectively in a single implant for contraception in breastfeeding women and shows promise for use in transdermal systems as a contraceptive or for HT when combined with estrogen.     

Sulfamoyloxy-substituted 2-phenylindoles: antiestrogen-based inhibitors of the steroid sulfatase in human breast cancer cells

Estrone sulfate (E1S) is an endogenous prodrug that delivers estrone and, subsequently, estradiol to the target cells following the hydrolysis by the enzyme estrone sulfatase which is active in various tissues including hormone dependent breast cancer cells. Blockade of this enzyme should reduce the estrogen level in breast cancer cells and prevent hormonal growth stimulation. Sulfamates of a variety of phenolic compounds have been shown to be inhibitors of estrone sulfatase. Our rational is based on findings that these inhibitors can undergo hydrolysis and the pharmacological effects of the free hydroxy compounds contribute to the bioactivity of the sulfamates. A desirable action of the metabolites would be an estrogen antagonism to block stimulatory effects of residual amounts of estrogens. Thus, we synthesized a number of sulfamoyloxy-substituted 2-phenylindoles with side chains at the indole nitrogen that guarantee antiestrogenic activity. All of the new sulfamates were studied for their inhibitory effects on the enzyme estrone sulfatase from human breast cancer cells and their (anti)hormonal activities in stably transfected human MCF-7/2a mammary carcinoma cells. The hormonal profile of the sulfamates was partly reflected by the properties of the corresponding hydroxy precursors. Some of the sulfamoylated antiestrogens strongly inhibited estrone sulfatase activity with IC₅₀ values in the submicromolar range. They were devoid of agonist activity and suppressed estrone sulfate-stimulated gene expression mainly by blocking the enzyme. Examples are the disulfamates of the indoles ZK 119, 010 and ZK 164, 015. Their IC₅₀s for sulfatase inhibition were 0.3 and 0.2 μM, respectively, and 50 and 80 nM, respectively, for the inhibition of E1S-stimulated luciferase expression in transfected MCF-7 cells. With some of the new sulfamates an additional direct antiestrogenic effect was noticed which might be due to a partial hydrolysis during incubation and would improve the growth inhibitory effect on estrogen-sensitive breast cancer cells.     

Menstrual cycles in rhesus monkeys (Macaca mulatta) are unaffected by a single dose of the anesthetics ketamine and xylazine administered during the midfollicular phase at laparoscopy

To identify an anesthetic regimen that produces more complete relaxation and analgesia than ketamine hydrochloride (Ketaset®) alone, a combination of ketamine (15 mg/kg body weight) and the hypnotic xylazine (Rompun®, 0.33 mg/kg) was evaluated. Since the desired experimental application required that the anesthetic not interfere with normal hormonal events during the menstrual cycle, this combination administered on day 6 of the cycle was tested to determine whether hormonal surges, incidence of ovulation, or cycle length would be altered relative to the use of ketamine alone. In five of six animals, ketamine plus xylazine had no effect on the occurrence of timely surges of estrogen, luteinizing hormone (LH), or follicle-stimulating hormone (FSH), or on ovulation as determined by the presence of a corpus luteum at laparoscopy and normal serum concentrations of progesterone. There were no significant differences between the cycle during treatment and previous cycles in the same animal for length of the menstrual cycle (26.0 ± 2.3 [5] days; X? ± S.D. [n] or luteal phase (13.4 ± 2.4 [5] days). Likewise, these values did not differ from those of ten control monkeys treated with ketumine only on day 5 or 6 of the cycle (incidence of ovulation, 10/10; cycle length, 27.9 ± 1.8 [10]; luteal phase length, 15.1 ± 1.4 [10], P > 0.05). Patterns of circulating progesterone were not altered by the addition of xylazine anesthesia. These findings indicate that xylazine, given in the midfollicular phase, did not alter ovulatory events or menstrual cycle characteristics in rhesus monkeys. Ketamine plus xylazine apparently provides anesthesia appropriate for laparoscopy.     

Morphologic responses of the mouse ovarian surface epithelium to ovulation and steroid hormonal milieu

Ovarian cancer of surface epithelial origin is an ovulation- and endocrine-related disease. It appears that a cell transformed by genotoxins generated at follicular rupture is propagated during postovulatory wound repair. A consequent steroid hormonal imbalance favoring the mitogenic estrogens is a prospective predisposing factor in ovarian neoplasia. Protection against epithelial ovarian cancer is conferred by progesterone. The objective of this study was to characterize the acute effects of ovulation and steroid hormonal exposure on morphologic responses of surface epithelial cells of mouse ovaries. Follicular development and ovulation were induced in immature animals with equine and human (=Day 0) choriogonadotropins, respectively. On Day 2 (approximately 36 hrs after ovulation), surface epithelial classifications presented in histologic sections were altered from simple (single-layered) squamous and cuboidal toward stratification; this trend was reversed (i.e., reverted to the control status) on Days 4-8. Shifts in the ovarian epithelium from simple to stratified were accentuated following postovulatory (Days 1-8) treatment with estradiol. Surface epithelia of ovaries obtained after 1 week of progesterone administration were exclusively of a simple phenotype. We conclude that the proliferative/procarcinogenic reaction of the ovarian surface epithelium to ovulation is exacerbated by estrogen and counteracted by progesterone.     

Aromatase and its inhibitors--an overview

Estrogen synthesis by aromatase occurs in a number of tissues throughout the body. Strategies which reduce production of estrogen offer useful means of treating hormone-dependent breast cancer. Initially, several steroidal compounds were determined to be selective inhibitors of aromatase. The most potent of these, 4-hydroxyandrostenedione (4-OHA) inhibits aromatase competitively but also causes inactivation of the enzyme. A number of other steroidal inhibitors appear to act by this mechanism also. In contrast, the newer imidazole compounds are reversible, competitive inhibitors. In vivo studies demonstrated that 4-OHA inhibited aromatase activity in ovarian and peripheral tissues and reduced plasma estrogen levels in rat and non-human primate species. In rats with mammary tumors, reduction in ovarian estrogen production was correlated with tumor regression. 4-OHA was also found to inhibit gonadotropin levels in animals in a dose-dependent manner. The mechanism of this effect appears to be associated with the weak androgenic activity of the compound. Together with aromatase inhibition, this action may contribute to reducing the growth stimulating effects of estrogen. A series of studies have now been completed in postmenopausal breast cancer patients treated with 4-OHA either 500 mg/2 weeks or weekly, or 250 mg/2 weeks. These doses did not affect gonadotropin levels. Plasma estrogen concentrations were significantly reduced. Complete or partial tumor regression occurred in 26% of the patients and the disease was stabilized in 25% of the patients. The results suggest that 4-OHA is of benefit to postmenopausal patients who have relapsed from prior hormonal therapies. Several of the steroidal inhibitors are now entering clinical trials as well as non-steroidal compounds which are more potent and selective than aminoglutethimide. Aromatase inhibitors should provide several useful additions to the treatment of breast cancer.     

Perceptual-motor performance during the menstrual cycle

Study I: Choice reaction times were measured in 12 normally menstruating women using a numerals-keys serial response task with three set sizes (two, four, and eight). Testing was carried out at four successive phases of the menstrual cycle corresponding with different states of neuroendocrine activity. They were, respectively: (i) 2nd day of flow; (ii) 4th day after cessation of flow (preovulation); (iii) 12th day after flow (postovulation); and (iv) 3rd day before next (estimated) flow. No consistent relationship between performance and phase of the cycle could be demonstrated. The only significant effect was an increase in reaction time with increasing set size. The study provides no support for the theory that variations in estrogen and progesterone levels may cause behaviorally relevant changes in central nervous system functioning. Study II: Four women suffering from premenstrual tension were examined thrice weekly on the same task for 4 consecutive weeks. Three hours prior to testing, a blood sample was drawn and assayed for estrogen, progesterone, and gonadotropin (LH). Performance on the serial choice response task bore no consistent relationship to blood levels of any of the three hormones. Performance before ovulation (as determined by peak LH level) did not differ significantly from that after ovulation. These results concur with those of Study I: there was no reliable relationship between hormonal status and performance.     

Relationships between ovarian morphology, vaginal cytology, serum progesterone, and urinary immunoreactive pregnanediol during the menstrual cycle of the cynomolgus monkey

Three different indices of ovulation and luteal activity were studied in eight regularly cycling cynomolgus monkeys. A significant relation between changes in serum progesterone and immunoreactive pregnanediol (I-PD) in urine was obtained. The occurrence of ovulation could be determined reliably from a change in the ratio of cornified to basal epithelial cells in vaginal smears, and luteal activity could be assessed reliably from daily measurements of urinary pregnanediol. The time of ovulation could be defined more precisely by daily I-PD radioimmunoassays than by the vaginal smear pattern. Measurements of I-PD also have the advantage of ease and noninvasiveness over serum progesterone determinations. More detailed information about changes in hormonal activities could not be obtained reproducibly from thorough examination of cell types in vaginal smears.     

Estrogen is not directly required for oocyte developmental competence

Oocyte maturation and ovulation require a coordinated interaction between gonadotrophs, steroid hormones, and growth factors. The extent to which estrogen is required in this process, however, remains unclear. To better understand the role of estrogen in maintaining developmental competence of mammalian oocytes, we studied the Aromatase knockout (ArKO) mouse, which has been genetically engineered to be incapable of synthesizing endogenous estrogen. Previous studies have established that ArKO female mice are anovulatory with ovaries that progressively degenerate, developing hemorrhagic cystic follicles. In young ArKO females, however, apparently healthy follicles and oocytes have been observed. We investigated if these oocytes could be induced to ovulate, then mature, fertilize, and develop in vitro. Following a standard superovulation protocol, ArKO oocytes did not ovulate. When recovered manually from the ovary, however, ArKO oocytes successfully progressed through in vitro maturation, fertilization, and development to the blastocyst stage at the same rate as wild-type and heterozygote littermates. Therefore, it appears that estrogen is not required for the production and growth of oocytes capable of maturation and complete preimplantation development but is required for continued follicle growth and feedback regulation of ovulation.