In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca(2+) channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca(2+) channel blocker, and chelation of extracellular Ca(2+). These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.

The comparative gonadal toxicity following two equally effective and non-cross-resistant regimens (MOPP and ABVD) was prospectively evaluated in 53 males with Hodgkin's disease. The median age was 29 yr (range 16-45). MOPP produced azoospermia in 28/29 patients (97%) while ABVD induced oligoazoospermia in 13/24 patients (54%). Follicle-stimulating hormone levels were consistently and significantly increased after MOPP while their median value remained within normal range after ABVD. Sperm count was repeated in 34 patients. Recovery of spermatogenesis occurred in 3/21 cases treated with MOPP and in all 13 cases given ABVD. Present findings confirm that the two alkylating agents, mechlorethamine and procarbazine, included in the MOPP regimen cause sterility in most patients while the drugs included in ABVD are not associated with permanent gonadal dysfunction.

A Mr 35,000 protein with follicle-stimulating hormone release-inhibitory activity was isolated from porcine ovarian follicular fluid by heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and multiple steps of high-performance liquid chromatography. The isolated molecule is highly enriched in cysteines and is composed of a single polypeptide chain. In addition, it has no sequence homology with the previously characterized follicular fluid inhibins, which are heterodimeric proteins of Mr 32,000 with follicle-stimulating hormone release-inhibiting activity. This protein specifically inhibits the basal secretion of follicle-stimulating hormone, but not that of luteinizing hormone, in the rat anterior pituitary monolayer culture system with a half-maximal effective dose of 2.5-6.0 ng/ml. Another form of the molecule of Mr 32,000 present in much lower concentration in follicular fluid was also isolated. It may differ from the Mr 35,000 form in glycosylation or carboxyl-terminal truncation. We suggest that this compound be called "follistatin" to signify its structural difference from inhibin.

In 2001, the Stages of Reproductive Aging Workshop (STRAW) proposed bleeding and endocrine criteria for defining the early and late menopausal transition stages. Based on expert consensus, STRAW recommended a shorter interval of amenorrhea than the commonly used 90-day amenorrhea criteria for late transition and a >7-day change in cycle length for early transition. The ReSTAGE collaboration used prospective menstrual calendar data from four cohorts (TREMIN, Melbourne Women's Midlife Health Project, Seattle Midlife Women's Health Study, and Study of Women's Health Across the Nation) to quantitatively evaluate STRAW's recommendations. This empirical assessment supported the STRAW recommendations that (1)>> or =60 days of amenorrhea be used to define the late menopausal transition and (2) that early transition is consistent with a persistent 7 or more day difference in length of consecutive cycles. Serum follicle stimulating hormone (FSH) values>> or =40 IU/l was an independent marker of the transition and, when occurring together with a bleeding marker, increased prediction of final menstrual period. Such a FSH criterion could be incorporated into the STRAW paradigm to facilitate prediction of proximity of the final menstrual period.

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.

Participation rates in epidemiologic studies on semen quality are generally very low, raising concerns as to the potential for selection bias. Since hormones both initiate and maintain spermatogenesis, they may serve as surrogates of semen quality in epidemiologic studies. For this reason, in the present study, we explored the influence and predictive ability of reproductive and thyroid hormones on semen quality among men who were partners in an infertile couple. Between 1999 and 2003, 388 men were recruited from Massachusetts General Hospital Andrology Laboratory for clinical evaluation of fertility status. Fresh semen samples were assessed for quality (concentration, motility and morphology) and the serum levels of hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin B, sex hormone-binding globulin (SHBG), testosterone, free androgen index, free T4, total T3, and thyroid-stimulating hormone (TSH), were measured. Multiple logistic regression revealed increased odds for below-reference sperm concentration and morphology in men with increased FSH, and decreased odds for below-reference sperm concentration and motility in men with increased inhibin B. When FSH and inhibin B were divided into quintiles, the relationships with sperm concentration showed evidence of a threshold value. However, the ability of specific FSH (10 IU/L) and/or inhibin B (80 pg/mL) cutoff values to predict semen quality was lower than in previous reports. In multiple linear regression analysis, FSH and LH were inversely associated with sperm concentration, motility, and morphology. Inhibin B and free T4 were positively associated with sperm concentration, while there was a suggestive positive association between testosterone and sperm motility. In conclusion, we have found that FSH, LH, inhibin B, testosterone and free T4 levels are associated with human semen parameters. Additional consideration should be given to the utility of serum hormone levels as a surrogate for semen quality in epidemiologic studies in which the collection of semen is difficult due to logistical and/or volunteer rate constraints.

Recovery of testicular spermatozoa from azoospermic patients with testicular failure followed by intracytoplasmic sperm injection (ICSI) is a recent advance in the treatment of male infertility. This study aimed at investigating which parameter(s) may predict successful testicular sperm recovery. We reviewed 395 testicular sperm recovery procedures and analysed the most frequently available parameters for clinical decision-making in azoospermic patients: (i) presence of at least one single spermatozoon in at least one preliminary semen analysis; (ii) maximum testicular volume; (iii) serum follicle stimulating hormone (FSH); and (iv) presence of spermatozoa in the histology of a randomly-taken testicular biopsy. Sensitivity, specificity, positive and negative predictive value, positive and negative likelihood ratio and accuracy were calculated for the above index parameters in different clinically relevant subgroups using receiver operating characteristic (ROC) curves whenever possible. Spermatozoa were always successfully recovered in patients with normal testicular histological findings (n = 173) or hypospermatogenesis (n = 16) but not in some patients with tubular sclerosis (seven out of 18), Sertoli cell-only pattern (55 out of 112) or maturation arrest (39 out of 76). Histopathology was the best test for predicting successful sperm recovery in the whole population (sensitivity: 86%, specificity: 93%, accuracy: 0.87). In patients with secretory azoospermia, histopathology was again the most accurate parameter (accuracy: 0.74), especially in patients showing Sertoli cell-only pattern (accuracy: 0.83) but not in patients showing maturation arrest (accuracy: 0.55). In patients with serum FSH concentrations>> 12 IU/l and maximum testicular volume < 15 ml, histopathology was not found to be accurate. Semen analysis, maximum testicular volume and serum FSH were not highly predictive in all subgroups studied. Our analysis shows that no strong predictors for successful testicular sperm recovery are available except for testicular histopathology.

The associations of body mass and body fat distribution, as measured by waist-to-hip circumference ratio, with serum concentrations of sex hormones and sex hormone binding globulin were examined in 88 postmenopausal women. Body mass index (BMI) was significantly and negatively associated with sex hormone binding globulin (SHBG) (r = -0.41), luteinizing hormone (LH) (r = -0.40) and follicle stimulating hormone (FSH) (r = -0.38) and was also significantly positively associated with both total and free oestradiol (r = 0.40 and 0.45, respectively). Waist-to-hip circumference ratio was significantly negatively correlated with SHBG (r = -0.53), LH (r = -0.35), and FSH (r = -0.35). After adjustment for BMI and other related factors, waist-to-hip circumference ratio was significantly and negatively associated with SHBG, LH, and FSH, and demonstrated a significant curvilinear relationship with free testosterone. These results suggest that in postmenopausal women, abdominal adiposity is associated with a relatively more androgenic sex hormone profile.

Secretion of luteinizing hormone in response to gonadotropin releasing hormone (GnRH) has been described in the recently developed LbetaT2 gonadotroph cell line. We evaluated the expression of follicle stimulating hormone (FSH)beta mRNA and secretion of FSH from LbetaT2 cells in response to GnRH and activin A. LbetaT2 cells were treated with activin A in doses from 0 to 50 ng/ml, with or without a daily 10 nM GnRH pulse, or with GnRH alone. FSH secretion was stimulated over 6-fold by concomitant GnRH and activin A in a dose-responsive fashion at 72 h of treatment. FSHbeta mRNA was detectable by ribonuclease protection assay only in cells treated with activin A with or without GnRH. The demonstration of FSHbeta gene expression in LbetaT2 cells further validates these cells as mature, differentiated gonadotrophs and as an important tool for the study of gonadotroph physiology.

The purpose of this study was to compare the midterm results of a radiological and surgical approach to uterine fibroids. One hundred twenty-one women with reproductive plans who presented with an intramural fibroid(s) larger than 4 cm were randomly selected for either uterine artery embolization (UAE) or myomectomy. We compared the efficacy and safety of the two procedures and their impact on patient fertility. Fifty-eight embolizations and 63 myomectomies (42 laparoscopic, 21 open) were performed. One hundred eighteen patients have finished at least a 12-month follow-up; the mean follow-up in the entire study population was 24.9 months. Embolized patients underwent a significantly shorter procedure and required a shorter hospital stay and recovery period. They also presented with a lower CRP concentration on the second day after the procedure (p < 0.0001 for all parameters). There were no significant differences between the two groups in the rate of technical success, symptomatic effectiveness, postprocedural follicle stimulating hormone levels, number of reinterventions for fibroid recurrence or regrowth, or complication rates. Forty women after myomectomy and 26 after UAE have tried to conceive, and of these we registered 50 gestations in 45 women. There were more pregnancies (33) and labors (19) and fewer abortions (6) after surgery than after embolization (17 pregnancies, 5 labors, 9 abortions) (p < 0.05). Obstetrical and perinatal results were similar in both groups, possibly due to the low number of labors after UAE to date. We conclude that UAE is less invasive and as symptomatically effective and safe as myomectomy, but myomectomy appears to have superior reproductive outcomes in the first 2 years after treatment.

Extending the FSH window for multifollicular development by administering FSH from the midfollicular phase onward constitutes a novel mild protocol for ovarian stimulation for in vitro fertilization (IVF) based on the physiology of single dominant follicle selection in normo-ovulatory women. We compared outcomes from this protocol with two other stimulation protocols. One hundred and forty-two normo-ovulatory patients with an indication for IVF (or IVF/ICSI) were randomized to a GnRH agonist long protocol (group A; n = 45) or one of two GnRH antagonist protocols commencing recombinant FSH on cycle d 2 (group B; n = 48) or cycle d 5 (group C; n = 49). A fixed dose (150 IU/d) of rFSH was used for ovarian stimulation, and GnRH antagonist cotreatment was initiated on the day when the leading follicle reached 14 mm diameter. Group C showed a shorter duration of stimulation (median duration, 11, 9, and 8 d for groups A, B, and C, respectively; P < 0.001), reflected in a significantly lower total dose of rFSH used (median amount of rFSH, 1650, 1350, and 1200 IU for groups A, B, and C, respectively; P < 0.001). In group C more cycles were cancelled during the stimulation phase due to insufficient response, resulting in a lower percentage of oocyte retrievals (84%, 73%, and 63% for groups A, B, and C; P = 0.02). However, women in group C obtained better quality embryos (percentage of embryo score of 1 for best embryo, 29%, 37%, and 61% for groups A, B, and C, respectively; P = 0.008), resulting in more transfers per oocyte retrieval (68%, 71%, and 90% for groups A, B, and C, respectively; P = 0.04). After profound ovarian stimulation (groups A and B) only 7% of the patients who retrieved four oocytes or less conceived, whereas after mild stimulation (group C) 67% of these patients conceived (P < 0.01). Overall, no differences were found among the three groups comparing pregnancy rate per started IVF cycle. In conclusion, application of the described mild ovarian stimulation protocol resulted in pregnancy rates per started IVF cycle similar to those observed after profound stimulation with GnRH agonist cotreatment despite shorter stimulation and a 27% reduction in exogenous FSH. A higher cancellation rate before oocyte retrieval was compensated by improved embryo quality concomitant with a higher chance of undergoing embryo transfer. A relatively low number of oocytes retrieved after mild ovarian stimulation distinctly differs from the pathological reduction in the number of oocytes retrieved after profound ovarian stimulation (poor response) associated with poor IVF outcome. The relatively small number of oocytes obtained after mild ovarian stimulation may represent the best of the cohort in a given cycle.

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.

OBJECTIVE

The aim of this article is to summarize the recommended updates to the 2001 Stages of Reproductive Aging Workshop (STRAW) criteria. The 2011 STRAW +10 reviewed advances in understanding of the critical changes in hypothalamic-pituitary-ovarian function that occur before and after the final menstrual period.

METHODS

Scientists from five countries and multiple disciplines evaluated data from cohort studies of midlife women and in the context of chronic illness and endocrine disorders on change in menstrual, endocrine, and ovarian markers of reproductive aging including antimüllerian hormone, inhibin-B, follicle-stimulating hormone, and antral follicle count. Modifications were adopted by consensus.

RESULTS

STRAW +10 simplified bleeding criteria for the early and late menopausal transition, recommended modifications to criteria for the late reproductive stage (Stage -3) and the early postmenopause stage (Stage +1), provided information on the duration of the late transition (Stage -1) and early postmenopause (Stage +1), and recommended application regardless of women's age, ethnicity, body size, or lifestyle characteristics.

CONCLUSIONS

STRAW +10 provides a more comprehensive basis for assessing reproductive aging in research and clinical contexts. Application of the STRAW +10 staging system should improve comparability of studies of midlife women and facilitate clinical decision making. Nonetheless, important knowledge gaps persist, and seven research priorities are identified.

Inactivation and silencing of the tumor suppressor PTEN are found in many different epithelial tumors, including thyroid neoplasia. Cowden Disease patients, who harbor germ-line PTEN mutations, often display thyroid abnormalities, including multinodular goiter and follicular adenomas, and are at increased risk of thyroid cancer. To gain insights into the role PTEN plays in thyroid function and disease, we have generated a mouse strain, in which Cre-mediated recombination is used to specifically delete Pten in the thyrocytes. We found that Pten mutant mice develop diffuse goiter characterized by extremely enlarged follicles, in the presence of normal thyroid-stimulating hormone and T4 hormone levels. Loss of Pten resulted in a significant increase in the thyrocyte proliferative index, which was more prominent in the female mice, and in increased cell density in the female thyroid glands. Surprisingly, goitrogen treatment did not cause a substantial increase of the mutant thyroid size and increased only to some extent the proliferation index of the female thyrocytes, suggesting that a relevant part of the thyroid-stimulating hormone-induced proliferation signals are funneled through the phosphatidylinositol-3-kinase (PI3K)/Akt cascade. Although complete loss of Pten was not sufficient to cause invasive tumors, over two thirds of the mutant females developed follicular adenomas by 10 months of age, showing that loss of Pten renders the thyroid highly susceptible to neoplastic transformation through mechanisms that include increased thyrocyte proliferation. Our findings show that constitutive activation of the PI3K/Akt cascade is sufficient to stimulate continuous autonomous growth and provide novel clues to the pathogenesis of Cowden Disease and sporadic nontoxic goiter.

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin-like G protein-coupled receptors with a large extracellular N-terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine-rich repeats (LRRs). The LRRs form a succession of beta-strands and alpha-helices organized into a horseshoe-shaped structure. It has been proposed that glycoprotein hormones interact with residues of the beta-strands making the concave surface of the horseshoe. Gain-of-function homology scanning of the beta-strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild-type LH/CGr. Molecular dynamics simulations and additional site-directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family.

Sox3 is expressed in developing gonads and in the brain. Evolutionary evidence suggests that the X-chromosomal Sox3 gene may be the ancestral precursor of Sry, a sex-determining gene, and Sox3 has been proposed to play a role in sex determination. However, patients with mutations in SOX3 exhibit normal gonadal determination but are mentally retarded and have short stature secondary to growth hormone (GH) deficiency. We used Cre-LoxP targeted mutagenesis to delete Sox3 from mice. Null mice of both sexes had no overt behavioral deficits and exhibited normal GH gene expression. Low body weight was observed for some mice; overgrowth and misalignment of the front teeth was observed consistently. Female Sox3 null mice (-/-) developed ovaries but had excess follicular atresia, ovulation of defective oocytes, and severely reduced fertility. Pituitary (luteinizing hormone and follicle-stimulating hormone) and uterine functions were normal in females. Hemizygous male null mice (-/Y) developed testes but were hypogonadal. Testis weight was reduced by 42%, and there was extensive Sertoli cell vacuolization, loss of germ cells, reduced sperm counts, and disruption of the seminiferous tubules. We conclude that Sox3 is not required for gonadal determination but is important for normal oocyte development and male testis differentiation and gametogenesis.

Leptin is a hormone involved in the regulation of body weight and sexual maturation. We previously reported that cancer cachexia was associated with reduced or normal levels of leptin. Here we investigate whether leptin levels are related to cachetic or hormonal status. Circulating leptin and its mRNA from adipose tissue were measured in 87 patients with gynaecological and breast cancers and related to tumour, cachexia and hormonal markers. We found that leptin protein increased in patients with these tumours due to higher mRNA levels. In patients with ovarian cancer, the increased leptin levels were associated with higher circulating follicle-stimulating hormone (FSH). The higher leptin concentrations in patients with endometrial and portio tumours were related to an increase in tissue estrogen receptor (ER) and progesterone receptor (PGR) and, only in the postmenopause, to an increase in circulating estradiol. Patients with breast cancer showed enhanced blood plasma concentrations of progesterone and estradiol, and enhanced tissue levels of ER and PGR associated with increased leptin levels. The data from the present study indicate that, in gynaecological and breast cancers, leptin is related to hormonal status but not to cachexia. We suggest that leptin stimulates the production of sexual hormones, important risk factors for these tumours, and we propose leptin as a novel prognostic marker.

OBJECTIVE

Chemotherapy-induced amenorrhea is a serious concern for women undergoing cancer therapy. This prospective randomized trial evaluated the use of gonadotropin-releasing hormone (GnRH) analog triptorelin to preserve ovarian function in women treated with chemotherapy for early-stage breast cancer.

METHODS

Premenopausal women age 44 years or younger were randomly assigned to receive either triptorelin or no triptorelin during (neo)adjuvant chemotherapy and were further stratified by age (< 35, 35 to 39,>> 39 years), estrogen receptor status, and chemotherapy regimen. Objectives included the resumption of menses and serial monitoring of follicle-stimulating hormone (FSH) and inhibin A and B levels.

RESULTS

Targeted for 124 patients with a planned 5-year follow-up, the trial was stopped for futility after 49 patients were enrolled (median age, 39 years; range, 21 to 43 years); 47 patients were treated according to assigned groups with four cycles of adriamycin plus cyclophosphamide alone or followed by four cycles of paclitaxel or six cycles of fluorouracil, epirubicin, and cyclophosphamide. Menstruation resumed in 19 (90%) of 21 patients in the control group and in 23 (88%) of 26 in the triptorelin group (P= .36). Menses returned after a median of 5.8 months (range, 1 to 19 months) after completion of chemotherapy in the triptorelin versus 5.0 months (range, 0 to 28 months) in the control arm (P= .58). Two patients (age 26 and 35 years at random assignment) in the control group had spontaneous pregnancies with term deliveries. FSH and inhibin B levels correlated with menstrual status.

CONCLUSIONS

When stratified for age, estrogen receptor status, and treatment regimen, amenorrhea rates on triptorelin were comparable to those seen in the control group.

The prevalence of neuropsychological and respiratory symptoms, lung ventilatory parameters, neurofunctional performances (visual reaction time, eye-hand coordination, hand steadiness, audioverbal short term memory), and several biological parameters (calcium, iron, luteinising hormone (LH), follicle stimulating hormone (FSH), and prolactin concentrations in serum, blood counts, manganese (Mn) concentration in blood and in urine) were examined in a group of workers (n = 92) exposed to MnO2 dust in a dry alkaline battery factory and a matched control group (n = 101). In the battery plant, the current exposure of the workers to airborne Mn was measured with personal samplers and amounted on average (geometric mean) to 215 and 948 micrograms Mn/m3 for respirable and total dust respectively. For each worker, the lifetime integrated exposure to respirable and total airborne Mn dust was also assessed. The geometric means of the Mn concentrations in blood (MnB) and in urine (MnU) were significantly higher in the Mn exposed group than in the control group (MnB 0.81 v 0.68 microgram/100 ml; MnU 0.84 v 0.09 microgram/g creatinine). On an individual basis, MnU and MnB were not related to various external exposure parameters (duration of exposure, current exposure, or lifetime integrated exposure to airborne Mn). On a group basis, a statistically significant association was found between MnU and current Mn concentrations in air. No appreciable difference between the exposed and the control workers was found with regard to the other biological measurements (calcium, LH, FSH, and prolactin in serum). Although the erythropoietic parameters and serum iron concentration were in the normal range for both groups, there was a statistically significant trend towards lower values in the Mn exposed workers. The prevalences of reported neuropsychological and respiratory symptoms, the lung function parameters, and the audioverbal short term memory scores did not differ between the control and exposed groups. The Mn workers, however, performed the other neurofunctional tests (visual reaction time, eye-hand coordination, hand steadiness) less satisfactorily than the control workers. For these tests, the prevalences of abnormal results were related to the lifetime integrated exposure to total and respirable Mn dust. On the basis of logistic regression analysis it may be inferred that an increased risk of peripheral tremor exists when the lifetime integrated exposure to Mn dust exceeds 3575 or 730 micrograms Mn/m3 x year for total and respirable dust respectively. The results clearly support a previous proposal by the authors to decrease the current time weighted average exposure to Mn dust.

Insulin stimulated hair follicle growth in a dose-dependent manner over the range of 0.01 to 100 micrograms/ml. Maximum rates of hair follicle growth were observed when follicles were maintained in medium containing 10 micrograms/ml insulin, which is supraphysiologic. Hair follicles maintained in the absence of insulin or at physiologic levels showed premature entry into a catagen-like state. Insulin-like growth factor (IGF)-I and -II had no significant effect on hair follicle growth when maintained in the presence of 10 micrograms/ml insulin. However, in the absence of insulin, both IGF-I (0.01-100 ng/ml) and IGF-II (0.01-100 ng/ml) stimulated hair follicle growth in a dose-dependent manner. IGF-I was more potent than either insulin or IGF-II, stimulating maximum rates of hair follicle growth at 10 ng/ml, whereas IGF-II gave maximum stimulation at 100 ng/ml. The rates of hair follicle growth stimulated by 10 ng/ml IGF-I were identical to those stimulated by 10 micrograms/ml insulin. IGF-II (100 ng/ml), however, was unable to stimulate hair follicle growth to the same extent as insulin. Both IGF-I (10 ng/ml) and IGF-II (100 ng/ml) were more potent than insulin at preventing hair follicles from entering into a catagen-like state. Growth hormone had no effect on hair follicle growth or morphology in the absence of insulin. These data suggest that in vitro IGF-I may be an important physiologic regulator of hair growth and possibly the hair growth cycle. Moreover, the removal of insulin from tissue culture medium may be a useful method of generating large numbers of catagen hair follicles for further in vitro studies.

Oocytes of varying stages of maturity were aspirated from follicles primed with either human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG) or a combination of follicle-stimulating hormone (FSH), hMG and hCG. Of the aspirated oocytes from 44 cycles, 74 were considered to be immature by virtue of morphologic characteristics of the oocytes and the degree of intercellular expansion of the associated cumular and membrana granulosa cells. After incubation periods of 22 to 35 hours in a Ham's F-10-based culture medium, these immature oocytes were inseminated with sperm donated by the patient's husband. Ultimately, 44 conceptuses were transferred to the respective uteri of 30 patients. Eight pregnancies were established as a result of these 30 transfers, two of which resulted from the transfer of only developed immature oocytes.

Nitric oxide (NO) synthase, the enzyme which converts arginine into citrulline plus NO, a highly active free radical, has been found in many neurons in the brain, including neurons in the hypothalamus. Our previous experiments showed that norepinephrine-induced prostaglandin E2 release from hypothalamic explants incubated in vitro is mediated by NO. Since the release of luteinizing hormone-releasing hormone (LHRH) is also driven by norepinephrine and prostaglandin E2, we hypothesized that NO might also control pulsatile release of LHRH in vivo, resulting in turn in pulsatile release of luteinizing hormone (LH). To ascertain the role of NO in control of pulsatile LH release in vivo, an inhibitor of NO synthase, NG-monomethyl-L-arginine (NMMA), was microinjected into the third cerebral ventricle (1 mg/5 microliters) of conscious castrate male rats at time 0 and 60 min later; blood samples were taken every 10 min during this period. NMMA blocked pulsatile LH release within 20 min, and plasma LH concentration declined further without pulses after the injection at 60 min. Pulsatile release of LH was not altered in diluent-injected controls. NMMA did not alter pulsatile release of follicle-stimulating hormone, which suggests that its release does not require NO. Incubation of medial basal hypothalami with norepinephrine (10 microM) induced an increase in LHRH release that was inhibited by NMMA (300 microM). NMMA alone did not alter basal LHRH release, whereas it was augmented by sodium nitroprusside (100 microM), which releases NO spontaneously. This augmentation was prevented by hemoglobin (2 micrograms/ml), which binds the NO released by nitroprusside. Our previous experiments showed that norepinephrine-induced release of prostaglandin E2 is mediated by NO. Nitric oxidergic neurons were visualized in the median eminence adjacent to the LHRH terminals. The combined in vivo and in vitro results indicate that the pulsatile release of LHRH induced by norepinephrine is brought about by alpha 1-adrenergic activation of NO synthase. NO then induces prostaglandin E2 release that activates exocytosis of LHRH secretory granules into the portal vessels to induce pulsatile LH release.

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.

Adrenal hypoplasia congenita (AHC) is an X-linked disorder that typically presents with adrenal insufficiency during infancy. Hypogonadotropic hypogonadism (HHG) has been identified as a component of this disorder in affected individuals who survive into childhood. Recently, AHC was shown to be caused by mutations in DAX-1, a protein that is structurally similar in its carboxyterminal region to orphan nuclear receptors. We studied two kindreds with clinical features of AHC and HHG. DAX-1 mutations were identified in both families. In the JW kindred, a single base deletion at nucleotide 1219 was accompanied by an additional base substitution that resulted in a frameshift mutation at codon 329 followed by premature termination. In the MH kindred, a GGAT duplication at codon 418 caused a frameshift that also resulted in truncation of DAX-1. Baseline luteinizing hormone (LIT), follicle-stimulating hormone (FSH), and free-alpha-subunit (FAS) levels were determined during 24 h of frequent (q10 min) venous sampling. In patient MH, baseline LH levels were low, but FAS levels were within the normal range. In contrast, in patient JW, the mean LH and FSH were within the normal range during baseline sampling, but LH secretion was erratic rather than showing typical pulses. FAS was apulsatile for much of the day, but a surge was seen over a 3-4-h period. Pulsatile gonadotropin releasing hormone (GnRH) (25 ng/kg) was administered every 2 h for 7 d to assess pituitary responsiveness to exogenous GnRH. MH did not exhibit a gonadotropin response to pulsatile GnRH. JW exhibited a normal response to the first pulse of GnRH, but there was no increase in FAS. In contrast to the priming effect of GnRH in GnRH-deficient patients with Kallmann syndrome, GnRH pulses caused minimal secretory responses of LH and no FAS responses in patient JW. The initial LH response in patient JW implies a deficiency in hypothalamic GnRH. On the other hand, the failure to respond to pulsatile GnRH is consistent with a pituitary defect in gonadotropin production. These two cases exemplify the phenotypic heterogeneity of AHC/HHG, and suggest that DAX-1 mutations impair gonadotropin production by acting at both the hypothalamic and pituitary levels.