Azoospermia is found in up to 10 to 20 per cent of the men who present to an infertility clinic. The main causes are testicular failure and ductal obstruction. Testicular biopsy remains the definitive test used to differentiate these 2 disorders. A retrospective study of 133 azoospermic men was performed to determine the accuracy and limitations of noninvasive variables in predicting testicular failure in an effort to limit the need for diagnostic testicular biopsy. Of 49 patients (37 per cent) with ductal obstruction a third had bilateral vasal agenesis. The remaining 84 azoospermic patients (63 per cent) had testicular failure. The results of the complete evaluation of these patients are described. Among the 101 patients with a testicular biopsy confirmed diagnosis there was a significant difference in testicular size (p less than 0.001), ejaculate volume (p less than 0.001) and serum follicle-stimulating hormone (p less than 0.001) between patients with testicular failure and those with ductal obstruction. The sensitivity and specificity of various parameters were determined. The best criteria to predict ductal obstruction preoperatively are a serum follicle-stimulating hormone level of less than 2 times greater than normal and the absence of bilateral testicular atrophy (100 per cent sensitivity and 71 per cent specificity). An algorithm for evaluation of the azoospermic patient is described such that all men with ductal obstruction and a minimal number with testicular failure undergo testicular biopsy.

Phthalates are high-production-volume synthetic chemicals with ubiquitous human exposures because of their use in plastics and other common consumer products. Recent epidemiologic evidence suggests that women have a unique exposure profile to phthalates, which raises concern about the potential health hazards posed by such exposures. Research in our laboratory examines how phthalates interact with the female reproductive system in animal models to provide insights into the potential health effects of these chemicals in women. Here we review our work and the work of others studying these mechanisms and propose a model for the ovarian action of di-(2-ethylhexyl) phthalate (DEHP). In vivo, DEHP (2 g/kg) causes decreased serum estradiol levels, prolonged estrous cycles, and no ovulations in adult, cycling rats. In vitro, monoethylhexyl phthalate (MEHP; the active metabolite of DEHP) decreases granulosa cell aromatase RNA message and protein levels in a dose-dependent manner. MEHP is unique among the phthalates in its suppression of aromatase and in its ability to activate peroxisome proliferator-activated receptors (PPARs). We hypothesize that MEHP activates the PPARs to suppress aromatase in the granulosa cell. MEHP-, PPAR alpha-, and PPAR gamma-specific ligands all similarly decreased estradiol production and RNA message levels of aromatase in vitro. Our model shows that MEHP acts on the granulosa cell by decreasing cAMP stimulated by follicle stimulating hormone and by activating the PPARs, which leads to decreased aromatase transcription. Thus, the environmental contaminant DEHP, through its metabolite MEHP, acts through a receptor-mediated signaling pathway to suppress estradiol production in the ovary, leading to anovulation.

OBJECTIVE

Examine age-adjusted odds and racial/ethnic differences in self-reported difficulties falling and staying asleep and early morning awakening in midlife women to determine whether difficulty sleeping increased with progression through the menopausal transition.

METHODS

Longitudinal analysis.

METHODS

Community-based.

METHODS

3,045 Caucasian, African American, Chinese, Japanese, and Hispanic women, aged 42-52 years and pre- or early peri-menopausal at baseline, participating in the Study of Women's Health Across the Nation (SWAN).

METHODS

None.

RESULTS

Self-reported number of nights of difficulty falling asleep, staying asleep, and early morning awakening during the previous 2 weeks were obtained at baseline and 7 annual assessments. Random effects logistic regression was used to model associations between each of the 3 sleep measures and the menopausal transition, defined by bleeding patterns, vasomotor symptoms (VMS), and estradiol (E2) and follicle stimulating hormone (FSH) serum levels. Adjusted odds ratios (ORs) for difficulty falling asleep and staying asleep increased through the menopausal transition, but decreased for early morning awakening from late perimenopause to postmenopause. Naturally and surgically postmenopausal women using hormones, compared with those who were not, generally had lower ORs for disturbed sleep. More frequent VMS were associated with higher ORs of each sleep difficulty. Decreasing E2 levels were associated with higher ORs of trouble falling and staying asleep, and increasing FSH levels were associated with higher ORs of trouble staying asleep. Racial/ethnic differences were found for staying asleep and early morning awakening.

CONCLUSIONS

Progression through the menopausal transition as indicated by 3 menopausal characteristics--symptoms, bleeding-defined stages, and endogenous hormone levels--is associated with self-reported sleep disturbances.

The demonstration that luteinizing hormone (LH) release from the pituitary is episodic rather than constant raises fundamental questions regarding the physiologic control of pulsatile LH secretion and its possible alteration in patients with gonadal disorders. To evaluate this mode of LH secretion, quantitative means of analyzing LH pulse amplitude, frequency, shape, and area were established and utilized to study normal subjects and patients with disorders of gonadotropin secretion. Similar patterns of LH secretion were observed in normal men, in women during the follicular phase of the menstrual cycle, and in patients with hyper- and hypogonadotropism, hirsuitism, and amenorrhea (mean pulse amplitude 39-179% from nadir to peak, frequency 2.7-3.9 secretory spikes/6 h). These observations suggested that the pattern of LH secretion is similar in both normal individuals and in those with a variety of pathologic conditions. By contrast, the pattern of pulsatile secretion appeared to differ in the following conditions. LH pulses of higher amplitude (333+/-170%) and lower frequency (1.6+/-0.24 SEM/6 h) characterized the secretory patterns of women during the luteal phase of the menstrual cycle, suggesting that gonadal steroids may modulate LH pulses. LH pulses of low amplitude (26+/-2.1%) and frequency (1.3+/-0.36/6 h) were observed in women with anorexia nervosa. Either integrated LH levels or a mean LH level determined from multiple samples provided a more accurate reflection of gonadotropin secretion than the use of single LH measurements. With multiple sampling over 6 h, it was possible to reduce the 95% confidence limit of LH estimates from +/-50-90 to +/-12%. This allowed normal subjects to be distinguished from patients with low or moderately elevated LH levels in whom gonadotropin levels in single samples were often in the "normal range."Several aspects of the physiologic control of pulsatile LH secretion were studied. The concordance of follicle-stimulating hormone (FSH) with LH pulses progressively increased as LH pulse height increased (P < 0.01) suggesting possible hypothalamic mediation of gonadotropin pulses. Measurement of the "apparent half-life" of LH after secretory spikes revealed half times of 34-233 min. It is likely that this variability was attributable to at least two phenomena: (a) constant low level LH secretion that continued after certain secretory episodes but not others; (b) variable mixing of newly secreted LH into at least two pools. The alpha adrenergic-blocking agents, chlorpromazine and phentolamine, failed to block LH secretory spikes at doses sufficient to result in a 30 mm drop in systolic blood pressure in normal men.

Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.

Ovulation is a precisely timed process by which a mature oocyte is released from an ovarian follicle. This process is initiated by the pituitary surge of luteinizing hormone (LH), is temporally associated with transcriptional regulation of numerous genes, and is presumed to involve the synthesis and/or activation of specific proteases that degrade the follicle wall. The progesterone receptor (PR), a nuclear receptor transcription factor, is induced in granulosa cells of preovulatory follicles in response to the LH surge and has been shown to be essential for ovulation, because mice lacking PR fail to ovulate and are infertile. Using these mice as a model in which to elucidate PR-regulated genes in the ovulation process, we show that the matrix metalloproteinases MMP-2 and MMP-9 are not targets of PR during ovulation. In contrast, two other proteases, ADAMTS-1 (A disintegrin and metalloproteinase with thrombospondin-like motifs) and cathepsin L (a lysosomal cysteine protease), are transcriptional targets of PR action. ADAMTS-1 is induced after LH stimulation in granulosa cells of preovulatory follicles and depends on PR. Cathepsin L is induced in granulosa cells of growing follicles by follicle-stimulating hormone, but the highest levels of cathepsin L mRNA occur in preovulatory follicles in response to LH in a PR-dependent manner. The identification of two regulated proteases in the ovary, together with their abnormal expression in anovulatory PR knockout mice, suggests that each plays a critical role in follicular rupture and represents a major advance in our understanding of the proteolytic events that control ovulation.

BACKGROUND

In adult humans, the follicle-stimulating hormone (FSH) receptor is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. It is minimally expressed by the endothelial cells of gonadal blood vessels.

METHODS

We used immunohistochemical and immunoblotting techniques involving four separate FSH-receptor-specific monoclonal antibodies that recognize different FSH receptor epitopes and in situ hybridization to detect FSH receptor in tissue samples from patients with a wide range of tumors. Immunoelectron microscopy was used to detect FSH receptor in mouse tumors.

RESULTS

In all 1336 patients examined, FSH receptor was expressed by endothelial cells in tumors of all grades, including early T1 tumors. The tumors were located in the prostate, breast, colon, pancreas, urinary bladder, kidney, lung, liver, stomach, testis, and ovary. In specimens obtained during surgery performed to remove tumors, the FSH receptor was not expressed in the normal tissues located more than 10 mm from the tumors. The tumor lymphatic vessels did not express FSH receptor. The endothelial cells that expressed FSH receptor were located at the periphery of the tumors in a layer that was approximately 10 mm thick; this layer extended both into and outside of the tumor. Immunoelectron microscopy in mice with xenograft tumors, after perfusion with anti–FSH-receptor antibodies coupled to colloidal gold, showed that the FSH receptor is exposed on the luminal endothelial surface and can bind and internalize circulating ligands.

CONCLUSIONS

FSH receptor is selectively expressed on the surface of the blood vessels of a wide range of tumors. (Funded by INSERM.).

The interactions of peptide and steroid hormone signaling cascades in the ovary are critical for follicular growth, ovulation, and luteinization. Although the pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play key regulatory roles, their actions are also dependent on other peptide signaling pathways, including those stimulated by insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-beta) family members (e.g., inhibin, activin, growth differentiation factor-9, bone morphogenic proteins), fibroblast growth factor, and Wnts (via Frizzled receptors). Each of these factors is expressed and acts in a cell-specific manner at defined stages of follicular growth. IGF-1, estrogen, and FSH comprise one major regulatory system. The Wnt/Frizzled pathways define other aspects relating to ovarian embryogenesis and possibly ovulation and luteinization. Likewise, the steroid receptors as well as orphan nuclear receptors and their ligands impact ovarian cell function. The importance of these multiple signaling cascades has been documented by targeted deletion of specific genes. For example, mice null for the LH-induced genes progesterone receptor (PR) and cyclo-oxygenase-2 (COX-2) fail to ovulate. Whereas PR appears to regulate the induction of novel proteases, COX-2 appears to regulate cumulus expansion. This review summarizes some new aspects of peptide and steroid hormone signaling in the rodent ovary.

Using polycystic ovary syndrome (PCOS) as a model of insulin resistance and hyperandrogenism, our specific aim was to assess the effect of Metformin on lipoproteins, sex hormones, gonadotropins, and blood pressure in 26 women with PCOS who were studied at baseline, received Metformin 1.5 g/d for 8 weeks, and were then restudied. None of the women had normal menstrual cycles, 100% had multiple subcapsular follicules by pelvic ultrasound, 90% were hirsute, and 85% had high free testosterone. Comparing post-Metformin versus baseline levels, the Quetelet Index (QI) decreased 1.5% (P = .04) and the waist to hip ratio (WHR) decreased 2.8% (P = .003). After covariance adjusting for changes in the QI and WHR, on Metformin the area under the insulin curve (IA) during oral glucose tolerance testing decreased 35% (P = .04), and the insulin area to glucose area ratio decreased 31% (P = .03). On Metformin, covariance-adjusted systolic blood pressure (SBP) decreased (P = .04) and apo A-1 increased (P = .05). On Metformin, with improvement in insulin sensitivity, there were sharp reductions in covariance-adjusted luteinizing hormone ([LH] P = .0007), total testosterone ([T] P = .0004), free T (P = .0001), androstenedione (P = .002), dehydroepiandrosterone sulfate ([DHEAS] P = .006), and the free androgen index ([FAI] P = .0005), with increments in follicle-stimulating hormone ([FSH] P = .04) and sex hormone-binding globulin ([SHBG] P = .04).(ABSTRACT TRUNCATED AT 250 WORDS)

Female mice lacking the gene encoding the prostaglandin (PG) E(2) receptor subtype EP(2) (EP(2)(-/-)) become pregnant and deliver their pups at term, but with a much reduced litter size. A decrease in ovulation number and a much reduced fertilization rate were observed in EP(2)(-/-) females without difference of the uterus to support implantation of wild-type embryos. Treatment with gonadotropins induced EP(2) mRNA expression in the cumulus cells of ovarian follicles of wild-type mice. The immature cumuli oophori from wild-type mice expanded in vitro in response to both follicle-stimulating hormone and PGE(2), but the response to PGE(2) was absent in those from EP(2)(-/-) mice. Cumulus expansion proceeded normally in preovulatory follicles but became abortive in a number of ovulated complexes in EP(2)(-/-) mice, indicating that EP(2) is involved in cumulus expansion in the oviduct in vivo. No difference in the fertilization rate between wild-type and EP(2)(-/-) mice was found in in vitro studies using cumulus-free oocytes. These results indicate that PGE(2) cooperates with gonadotropin to complete cumulus expansion for successful fertilization.

The first inductive interaction in amphibian development is mesoderm induction, when a signal from the vegetal hemisphere of the blastula induces mesoderm from overlying equatorial cells. Recently, several 'mesoderm-inducing factors' (MIFs) have been discovered. These cause isolated Xenopus animal caps to form mesodermal cell types such as muscle, instead of their normal fate of epidermis. The MIFs fall into two classes. One comprises members of the fibroblast growth factor (FGF) family, and the other members of the transforming growth factor type beta (TGF-beta) family. Of the latter group, the most potent is XTC-MIF, a protein produced by Xenopus XTC cells. Here we show that XTC-MIF is the homologue of mammalian activin A. Activins modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and cause the differentiation of two erythroleukaemia cell lines. Our results indicate that these molecules may also act in early development during formation of the mesoderm.

BACKGROUND

Idiopathic hypogonadotropic hypogonadism, which may be associated with anosmia (the Kallmann syndrome) or with a normal sense of smell, is a treatable form of male infertility caused by a congenital defect in the secretion or action of gonadotropin-releasing hormone (GnRH). Patients have absent or incomplete sexual maturation by the age of 18. Idiopathic hypogonadotropic hypogonadism was previously thought to require lifelong therapy. We describe 15 men in whom reversal of idiopathic hypogonadotropic hypogonadism was sustained after discontinuation of hormonal therapy.

METHODS

We defined the sustained reversal of idiopathic hypogonadotropic hypogonadism as the presence of normal adult testosterone levels after hormonal therapy was discontinued.

RESULTS

Ten sustained reversals were identified retrospectively. Five sustained reversals were identified prospectively among 50 men with idiopathic hypogonadotropic hypogonadism after a mean (+/-SD) duration of treatment interruption of 6+/-3 weeks. Of the 15 men who had a sustained reversal, 4 had anosmia. At initial evaluation, 6 men had absent puberty, 9 had partial puberty, and all had abnormal secretion of GnRH-induced luteinizing hormone. All 15 men had received previous hormonal therapy to induce virilization, fertility, or both. Among those whose hypogonadism was reversed, the mean serum level of endogenous testosterone increased from 55+/-29 ng per deciliter (1.9+/-1.0 nmol per liter) to 386+/-91 ng per deciliter (13.4+/-3.2 nmol per liter, P<0.001), the luteinizing hormone level increased from 2.7+/-2.0 to 8.5+/-4.6 IU per liter (P<0.001), the level of follicle-stimulating hormone increased from 2.5+/-1.7 to 9.5+/-12.2 IU per liter (P<0.01), and testicular volume increased from 8+/-5 to 16+/-7 ml (P<0.001). Pulsatile luteinizing hormone secretion and spermatogenesis were documented.

CONCLUSIONS

Sustained reversal of normosmic idiopathic hypogonadotropic hypogonadism and the Kallmann syndrome was noted after discontinuation of treatment in about 10% of patients with either absent or partial puberty. Therefore, brief discontinuation of hormonal therapy to assess reversibility of hypogonadotropic hypogonadism is reasonable. (ClinicalTrials.gov number, NCT00392756 [ClinicalTrials.gov].).

We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.

The criteria for the diagnosis of the polycystic ovary syndrome (PCOS) have still not been agreed universally. A population of 1741 women with PCOS were studied, all of whom had polycystic ovaries seen by ultrasound scan. The frequency distributions of the serum concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone and prolactin and the body mass index, ovarian volume, uterine cross-sectional area and endometrial thickness were determined and compared with the symptoms and signs of PCOS. Obesity was associated with hirsutism and an elevated serum testosterone concentration and was also correlated with increased rates of infertility and cycle disturbance. The rates of infertility and cycle disturbance also increased with serum LH concentrations>> 10 IU/l. A rising serum concentration of testosterone [mean and 95th percentiles 2.6 (1.1-4.8) nmol/l] was associated with an increased risk of hirsutism, infertility and cycle disturbance. The ovarian volume was correlated with serum concentrations of testosterone, LH and the body mass index, which was also correlated with the uterine area. This descriptive data from the largest reported series of women with PCOS enables the development of a management-orientated approach to the syndrome. Women who are overweight can expect an improvement in their symptoms if they lose weight. An elevated concentration of LH >> 10 IU/l) is associated with infertility and treatment should be chosen accordingly. If the serum testosterone concentration is>> 4.8 nmol/l, other causes of hyperandrogenism should be excluded.

OBJECTIVE

To test the hypothesis that men with varicocele who have already fathered children are immune to the detrimental effect of varicocele on their fertility and will continue to be fertile. If this were the case, one would expect a very low incidence of varicocele in currently infertile men who were able to father a child in the past (secondary infertility) compared with men who have never been fertile (primary infertility).

METHODS

Survey of men with male factor infertility.

METHODS

Tertiary care university medical center.

METHODS

One thousand ninety-nine infertile men of whom 98 (9%) met our criteria for secondary infertility. Men with prior vasectomy and men whose partners were over age 40 were excluded.

METHODS

Difference in the incidence of varicocele in men with secondary infertility versus primary infertility.

RESULTS

A varicocele was palpable in 35% (352/1,001) of men with primary infertility and 81% (79/98) of men with secondary infertility. This difference in the incidence of varicocele was highly significant. Men with secondary infertility and varicocele were slightly older (37.9 versus 33.5 years), had a lower mean sperm concentration (30.2 versus 46.1 x 10(6)/mL), more abnormally shaped sperm (72% versus 40%), and higher mean serum follicle-stimulating hormone levels (17.6 versus 7.9 mIU/mL,) compared with men with primary infertility and varicocele.

CONCLUSIONS

The incidence of varicocele is much higher in male factor secondary infertility compared with primary infertility. These findings suggest that varicocele causes a progressive decline in fertility and that prior fertility in men with varicocele does not predict resistance to varicocele induced impairment of spermatogenesis. Men with a varicocele may benefit from early evaluation and prophylactic varicocelectomy to prevent future infertility.

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) act on gonadal cells to promote steroidogenesis and gametogenesis. Clarifying the in vivo roles of LH and FSH permits a feasible approach to contraception involving selective blockade of gonadotropin action. One way to address these physiologically important problems is to generate mice with an isolated LH deficiency and compare them with existing FSH loss-of-function mice. To model human reproductive disorders involving loss of LH function and to define LH-responsive genes, we produced knockout mice lacking the hormone-specific LHbeta-subunit. LHbeta-null mice are viable but demonstrate postnatal defects in gonadal growth and function resulting in infertility. Mutant males have decreased testes size, prominent Leydig cell hypoplasia, defects in expression of genes encoding steroid biosynthesis pathway enzymes, and reduced testosterone levels. Furthermore, spermatogenesis is blocked at the round spermatid stage, causing a total absence of the elongated spermatids. Mutant female mice are hypogonadal and demonstrate decreased levels of serum estradiol and progesterone. Ovarian histology demonstrates normal thecal layer, defects in folliculogenesis including many degenerating antral follicles, and absence of corpora lutea. The defects in both sexes are not secondary to aberrant FSH regulation, because FSH levels were unaffected in null mice. Finally, both male and female null mice can be pharmacologically rescued by exogenous human chorionic gonadotropin, indicating that LH-responsiveness of the target cells is not irreversibly lost. Thus, LHbeta null mice represent a model to study the consequences of an isolated deficiency of LH ligand in reproduction, while retaining normal LH-responsiveness in target cells.

We have previously shown that androgens stimulate early stages of follicular development and that granulosal androgen receptor (AR) gene expression is positively correlated with follicular growth. The present study was aimed at elucidating potential interactions between FSH and androgens in follicular development. Study groups included eight normal cycling rhesus monkeys (five follicular and three luteal-phase), eight testosterone (T)-treated, and four FSH-treated animals. Examination of sequential ovary sections revealed selective colocalization of AR and FSH receptor (FSHR) messenger RNAs (mRNAs) in healthy, growing follicles. Moreover, individual follicles demonstrate a highly significant (P < 0.001) positive correlation between FSHR and AR mRNA levels in all study groups. Androgen treatment significantly increased granulosa cell FSHR mRNA abundance (by approximately 50-100%, depending on follicle size). FSH treatment increased granulosa AR mRNA levels only in primary follicles. The finding that T augments follicular FSHR expression suggests that androgens promote follicular growth and estrogen biosynthesis indirectly, by amplifying FSH effect, and may partially explain the enhanced responsiveness to gonadotropin stimulation noted in women with polycystic ovary syndrome.

Pituitary adenomas may remain intrasellar or infiltrate dura and bone. Invasive adenomas are not considered to be malignant; in biological behavior they are between non-infiltrative adenomas and pituitary carcinomas. The latter are defined as tumors with subarachnoid, brain, or systemic metastasis. Invasion may be defined radiologically, operatively, or histologically. On the basis of operatively assessed tumor size and gross invasion of dura and bone as well as immunocytochemical and ultrastructural analysis of 365 pituitary adenomas, the following data were obtained. There were 23 growth hormone (GH)-cell adenomas: 14% microadenomas and 86% macroadenomas; their overall frequency of invasion was 50%. There were 24 prolactin (PRL)-cell adenomas: 33% microadenomas and 67% macroadenomas, with an overall frequency of invasion of 52%. Mixed GH-cell and PRL-cell adenomas were found in 35 cases; 26% were microadenomas and 74% were macroadenomas, and the overall frequency of invasion was 31%. Sixty patients had adrenocorticotropic hormone (ACTH)-cell adenomas (Cushing's disease): 87% microadenomas and 13% macroadenomas; the overall frequency of invasion was 25% (in 8% of microadenomas and 62% of macroadenomas). Twenty patients had ACTH-cell adenomas (Nelson's syndrome): 30% microadenomas and 70% macroadenomas; the overall frequency of invasion in these cases was 50% (in 17% of microadenomas and 64% of macroadenomas). Silent ACTH-cell adenomas, 100% macroadenomas, were found in 11 patients, with an 82% frequency of invasion. There were 32 follicle-stimulating and luteinizing hormone adenomas, all macroadenomas, with a frequency of invasion of 21%. Four patients had thyroid-stimulating hormone adenomas, all macroadenomas, with a 75% frequency of invasion. Null-cell adenomas were found in 93 cases: 2% microadenomas and 98% macroadenomas, with a frequency of invasion of 42%. There were 63 plurihormonal adenomas (GH, PRL, glycoprotein): 25% microadenomas and 75% macroadenomas, with a 50% overall frequency of invasion. Based on this study, and on their usual frequency of occurrence, the estimated rate of gross invasion by pituitary adenomas of all types is approximately 35%. It is concluded that immunocytochemical and ultrastructural characteristics of pituitary adenomas reflect the tendency of these tumors to infiltrate and hence may be of prognostic significance.

The melanogenic actions of the melanocortins are mediated by the melanocortin-1 receptor (MC1R). MC1R is a member of the G-protein-coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or alpha-melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist-independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.

Paracrine factors secreted by oocytes and somatic cells regulate many important aspects of early ovarian follicle development in mammals. From activation of dormant primordial follicles to selection of secondary follicles, locally acting factors have been identified that appear to exert important effects on the growth and differentiation of oocytes and granulosa cells. This article summarizes evidence to support a model for bi-directional paracrine communication that is based on developmental regulation of the delivery and reception of paracrine factors at the oocyte-granulosa cell interface. Transzonal projections that originate from granulosa cells and terminate at the oocyte plasma membrane provide a polarized means to orient the secretory organelles of somatic cells. Characterization of transzonal projections in follicles from normal and genetically modified mice reveals dynamic changes in the density and stability of transzonal projections. On the basis of new data analysing the orientation and cytoskeletal content of transzonal projections in mammalian oocytes, a model is proposed for regulation of paracrine growth factor secretion by follicle-stimulating hormone. These findings have immediate implications for ovarian hyperstimulation protocols and follicle culture models as related to the production of mammalian embryos by assisted reproductive technologies.

Cortical slices were prepared from the right ovaries of six lambs and either grafted directly to the ovarian pedicles of origin or cooled slowly to liquid nitrogen temperatures in medium containing dimethylsulphoxide. Three weeks later, the contra-lateral ovary was removed and replaced with frozen-thawed slices from the same animal. Two of the animals mated during their second oestrous cycle 3-4 months later and the remainder had at least one ovulatory cycle. The pregnancies reached full-term development, one lamb being derived from an ovulation in a fresh graft and the other from a frozen-thawed graft. None of the sheep had peripheral plasma concentrations of follicle stimulating hormone or luteinizing hormone consistently in the castrate range, and only one graft was devoid of follicles when the animals were slaughtered 9 months after the operations. Grafts with primordial follicles always contained developing follicles, which occasionally attained pre-ovulatory sizes of 7 mm in diameter. A corpus albicans was present in five grafts. Since all developing follicles had degenerated 1 week after grafting in an additional ewe, the large follicles in long-term grafts had presumably commenced growing after the operation. There were no obvious differences between fresh and frozen-thawed grafts in either appearance or weight, and all had apparently grown since implantation. Despite substantial depletion of primordial follicle numbers, the results indicated that frozen storage and replacement of a patient's own ovarian tissue might be practicable when fertility potential is threatened by chemotherapy/radiotherapy.

A defect in the structure of the obese gene is responsible for development of obesity in the ob/ob mouse. The product of expression of the gene is the protein hormone leptin. Leptin causes weight loss in ob/ob and normal mice, it is secreted by adipocytes, and it is an important controller of the size of fat stores by inhibiting appetite. The ob/ob mouse is infertile and has a pattern of gonadotropin secretion similar to that of prepubertal animals. Consequently, we hypothesized that leptin might play a role in the control of gonadotropin secretion and initiated studies on its possible acute effects on hypothalamic-pituitary function. After a preincubation period, hemi-anterior pituitaries of adult male rats were incubated with leptin for 3 hr. Leptin produced a dose-related increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) release, which reached peaks with 10(-9) and 10(-11) M leptin, respectively. Gonadotropin release decreased at higher concentrations of leptin to values indistinguishable from that of control pituitaries. On the other hand, prolactin secretion was greatly increased in a dose-related manner but only with leptin concentrations (10(-7)-10(-5) M). Incubation with leptin of median eminence-arcuate nuclear explants from the same animals produced significant increases in LH-releasing hormone (LHRH) release only at the lowest concentrations tested (10(-12)-10(-10) M). As the leptin concentration was increased, LHRH release decreased and was significantly less than control release at the highest concentration tested (10(-6) M). To determine if leptin can also release gonadotropins in vivo, ovariectomized females bearing implanted third ventricle cannulae were injected with 10 microg of estradiol benzoate s.c., followed 72 hr later by microinjection into the third ventricle of leptin (0.6 nmol in 5 microl) or an equal volume of diluent. There was a highly significant increase in plasma LH, which peaked 10-50 min after injection of leptin. Leptin had no effect on plasma FSH concentrations, and the diluent had no effect on either plasma FSH or LH. Thus, leptin at very low concentrations stimulated LHRH release from hypothalamic explants and FSH and LH release from anterior pituitaries of adult male rats in vitro and released LH, but not FSH, in vivo. The results indicate that leptin plays an important role in controlling gonadotropin secretion by stimulatory hypothalamic and pituitary actions.

FSH is secreted by gonadotropes of the anterior pituitary and plays a crucial role in mammalian reproduction. However, little is known about FSH gene regulation due to the lack of a gonadotrope cell line that synthesizes FSH. The LbetaT2 mouse pituitary cell line, isolated by targeted tumorigenesis in transgenic mice, has the characteristics of a mature gonadotrope, including expression of GnRH receptor, steroidogenic factor 1, and both the alpha- and beta-subunits of LH, but was thought not to express FSH. Using RT-PCR, we show that these cells synthesize FSH beta- subunit messenger RNA, which is induced by activin and inhibited by follistatin. Furthermore, in transient transfections an ovine FSHbeta 5'-regulatory region (5.5 kb) confers LbetaT2 cell-specific expression to a reporter gene compared with other pituitary and nonpituitary cell lines. This FSHbeta regulatory region responds to activin specifically in LbetaT2 cells, an effect that is blocked by follistatin. The LHbeta, alpha-subunit, and GnRH receptor regulatory regions are induced by activin and blocked by follistatin. Furthermore, LbetaT2 cells express the components of the activin system, and addition of follistatin alone reduces FSHbeta gene expression, demonstrating that an endogenous activin autocrine loop regulates FSH in these cells. In addition, GnRH stimulates both the FSHbeta and LHbeta regulatory regions, specifically in LbetaT2 cells. Surprisingly, GnRH induction is reduced by follistatin, suggesting its dependence on endogenous activin. As the mouse GnRH receptor promoter is inhibited by follistatin, reduction of GnRH receptor levels might be one mechanism by which follistatin interferes with GnRH induction of gonadotropin genes. In summary, LbetaT2 cells exhibit the characteristics of fully differentiated gonadotropes, including the expression of LH, FSH, GnRH receptor, and components of the activin/follistatin system, as well as display the appropriate responses to activin and GNRH:

To investigate further brain-pituitary-gonadal interrelationships we have generated mice in which the gene encoding the FSH receptor has been disrupted. Female FSH receptor knockout (FSHRKO) mice were infertile. The ovaries were significantly reduced in size, with follicular development arrested at the preantral stage, but there was evidence of stromal hypertrophy. The vagina was imperforate, and the uterus was atrophic. There was no response to administration of PMSG. Inhibins A and B were undetectable in both the serum and gonads. Compared with those in control animals, serum concentrations of FSH and LH were significantly elevated in mutant females. The pituitary content of FSH, but not LH, was also significantly elevated. Estrogen administration in FSHRKO female mice suppressed serum LH levels to those seen in control mice, whereas FSH levels were reduced by only 50%. Male FSHRKO mice were fertile, although testis weight was significantly reduced. However, testicular inhibin A and B concentrations did not differ from those in normal littermates. Serum levels of FSH and LH were elevated in the null mutant male mice, whereas no differences were found in the pituitary content of these hormones. In conclusion, ovarian follicular development cannot progress beyond the preantral stage without FSH. In the absence of mature follicles ovarian estrogen remains low, and consequently accessory sex tissue growth and negative feedback regulation of gonadotropin secretion are severely compromised. In the male, however, inability to respond to FSH does not impair fertility, although testicular weight is reduced, and feedback regulation of pituitary gonadotropins and intratesticular paracrine interactions may be disturbed.