A novel reproductive peptide, phoenixin.

Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones luteinising hormone and follicle-stimulating hormone is regulated primarily by hypothalamically-derived gonadotrophin-releasing hormone (GnRH), although other releasing factors (RF) have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotrophin secretion by modulating the expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using small interfering RNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretary vesicles to pituitary membranes.

Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells.

In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P < 0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P < 0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P < 0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P > 0.10). Treatment with 600 ng/ml GDF-9 increased (P < 0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P < 0.05) progesterone production (by 28%), increased (P < 0.05) cell numbers (by 60%), and had no effect (P > 0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P < 0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.

Hormonology: a genomic perspective on hormonal research.

Recent advances in comparative genomics allow a new paradigm for hormonal research. At the centennial of the first use of the term hormone by Ernest Starling, we reflected on the changing approaches in elucidating hormonal signaling mechanisms and highlighted the inadequacy of the term endocrinology, implying remote activation, to describe the diverse modes of hormone actions. Several examples were presented to underscore the power of comparative genomics in the identification of new polypeptide hormones, receptors, and signaling pathways. We propose the use of the term hormonology to more accurately reflect the expanding boundaries of the discipline.

Protective effects of the urocortin homologues stresscopin (SCP) and stresscopin-related peptide (SRP) against hypoxia/reoxygenation injury in rat neonatal cardiomyocytes.

Urocortin (UCN), a member of the Corticotropin-Releasing Factor (CRF) family of peptides is a well described cardioprotective agent. UCN is able to bind to two types of G-protein coupled receptors: CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2), whereas, two homologues of UCN, stresscopin (SCP) or also known as urocortin III (UCNIII) and stresscopin related peptide (SRP), or urocortin II (UCNII), bind exclusively and with high affinity to CRFR2, we hypothesised that they will exhibit more pronounced cardioprotective effects than UCN. We show for the first time that SCP is expressed in rat cardiomyocytes and that the levels of SRP and SCP are increased by hypoxic stress. All three peptides have potent cardioprotective effects in cells exposed to hypoxia/reoxygenation. When used at 10(-8) M they increased the amount of live cells by 25% when added prior to hypoxia, and by 20% when UCN and SCP were added at the onset of reoxygenation. In addition, the peptides are equally are more potent antiapoptotic factors than UCN. The antiapoptotic effects of SCP were more pronounced than SRP and UCN at a concentration of 10(-10) M. Furthermore, SCP and SRP protect cardiomyocytes better than UCN at concentrations up to and including 10(-10) M and reduced the amount of TUNEL positive cells almost by half at concentrations of 10(-12) to 10(-10) M. More importantly, we demonstrate that SCP and SRP are able to protect cardiomyocytes even if they are administered after the hypoxic insult and prior to reoxygenation. In this case SCP was more potent than UCN and SRP at 10(-12) M and both SCP and SRP exhibited higher protection at 10(-8) M compared to UCN. Cardioprotection of cardiomyocytes by 10(-8) M of peptides was abolished when treated with 50 microM LY294002 or 100 microM PD98059, but not by 10 microM SB203580 prior to the hypoxic insult. Transfection of dominant negative Akt and MEK1 also blocked protection by the peptides, whereas dominant negative MEKK6 had no effects, demonstrating that SCP and SRP, like UCN, require activation of p42/44 Mitogen activated protein kinase and Akt/Protein Kinase B in order to produce their cardioprotective effects. In addition, we showed that SCP and UCN are potent activators of the p42/44 MAPK pathway, with SRP able to induce phosphorylation of p42/44 MAPK as well, albeit not as pronounced.

Growth differentiation factor-9 signaling in the ovary.

Growth differentiation factor-9 (GDF-9) is an oocyte-derived growth factor and a member of the transforming growth factor-beta (TGF-beta) superfamily. In GDF-9 null mice, follicle development is arrested at the primary stage and in vivo treatment with GDF-9 enhances the progression of primordial and primary follicles into small preantral follicles. In vitro, GDF-9 promotes granulosa cell proliferation but inhibits FSH-induced differentiation. GDF-9 also promotes the differentiation of theca cells in vivo and in vitro. GDF-9, like TGF-beta or activin, is a close member of the bone morphogenetic proteins (BMPs) family. GDF-9 likely initiates signaling by assembling two related but distinct types of receptors, both of which are serine/threonine kinases with a single transmembrane domain. The ligand-receptor binding activates intracellular transcription factors called Smads. In granulosa cells, Vitt et al. have shown that the BMP receptor type II is involved in GDF-9 signaling. The type I receptors and the Smad pathway for GDF-9 remain to be identified.

Impairment of spermatogenesis in transgenic mice with selective overexpression of Bcl-2 in the somatic cells of the testis.

To explore the functional role of Bcl-2 in germ cell development, transgenic mice carrying 6 kilobases of the inhibin-alpha promoter were generated to express human bcl-2 gene product in the gonads. Although female transgenic mice demonstrated decreased follicle apoptosis, enhanced folliculogenesis, and increased germ cell tumorigenesis, the adult males exhibited variable impairment of spermatogenesis. The degree of damage ranged from tubules with intraepithelial vacuoles of varying sizes to near atrophied tubules consisting of Sertoli cells and a few spermatogonia. Although there was no significant change in body weight, an approximately 34% decrease in testicular weights was noted in transgenic animals compared with wild-type mice. Gamete maturation, assessed by determining the percentage of tubules with advanced (steps 13-16) spermatids, was decreased to 44.4% of the values measured in the wild-type animals. The incidence of germ cell apoptosis increased 3.8-fold in the transgenic animals and was associated with a marked loss of germ cells. Electron microscopy of the testes further revealed large vacuoles in the Sertoli cell cytoplasm and dilations of the intracellular spaces between adjacent Sertoli cells, spermatid malformations, and increased germ cell apoptosis in the transgenic animals. There was no evidence of Sertoli cell death either by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay or electron microscopy. Leydig cell ultrastructure, cell size and numbers, and plasma levels of testosterone were not different between normal and the transgenic animals. Collectively, these results support the critical role of Bcl-2 in male germ cell development and are consistent with the gender-specific role of the Bcl-2 family members in reproduction.

Stage-dependent role of growth differentiation factor-9 in ovarian follicle development.

GDF-9 was shown to be essential for follicle progression and is the only factor secreted by the oocyte shown to increase the number of primordial and primary follicles in vivo. Furthermore, GDF-9 is a major growth factor involved in the oocyte control of granulosa cell differentiation. A concentration gradient of the paracrine factor GDF-9 established by the oocyte could provide the basis to explain the stratification of granulosa cells in antral and preovulatory follicles. The stimulatory effects of GDF-9 on early follicle development provide a basis for the use of GDF-9 in the treatment of infertility.

Underphosphorylated BAD interacts with diverse antiapoptotic Bcl-2 family proteins to regulate apoptosis.

Survival factors activate kinases which, in turn, phosphorylate the proapoptotic Bcl-xl/Bcl-2-associated death promoter homolog (BAD) protein at key serine residues. Phosphorylated BAD interacts with 14-3-3 proteins, and overexpression of 14-3-3 attenuates BAD-mediated apoptosis. Although BAD is known to interact with Bcl-2, Bcl-w, and Bcl-xL, the exact relationship between BAD and anti- or proapoptotic Bcl-2 proteins has not been analyzed systematically. Using the yeast two-hybrid protein interaction assay, we found that BAD interacted negligibly with proapoptotic Bcl-2 proteins. Even though wild type BAD only interacted with selected numbers of antiapoptotic proteins, underphosphorylated mutant BAD interacted with all antiapoptotic Bcl-2 proteins tested (Bcl-2, Bcl-w, Bcl-xL, Bfl-1/A1, Mcl-1, Ced-9, and BHRF-1). Using nonphosphorylated recombinant BAD expressed in bacteria, direct interactions between BAD and diverse antiapoptotic Bcl-2 members were also observed. Furthermore, apoptosis induced by BAD was blocked by coexpression with Bcl-2, Bcl-w, and Bfl-1. Comparison of BAD orthologs from zebrafish to human indicated the conservation of a 14-3-3 binding site and the BH3 domain during evolution. Thus, highly conserved BAD interacts with diverse antiapoptotic Bcl-2 members to regulate apoptosis.

DNA array analysis of changes in preovulatory gene expression in the rat ovary.

During the periovulatory period, the mammalian ovary is the site of dramatic functional and structural changes, leading to oocyte maturation, follicle rupture, and corpus luteum formation. To a large extent, these processes result from changes in the transcriptome of various ovarian cell types. To develop a broader view of periovulatory changes in gene expression in the ovary and to identify further genes involved in periovulatory events, we used the recently developed DNA array technology. Immature female eCG-primed rats were killed either immediately before or 6 h after ovulation induction with hCG. Total ovarian RNA was isolated and used to prepare radiolabeled cDNA probes, which were hybridized to DNA arrays representing approximately 600 rat genes. Quantitative analysis identified a multitude of regulated gene messages, including several genes involved in extracellular matrix degradation and lipid/steroid metabolism previously reported to be induced by hCG. This screening also identified a group of candidate genes whose ovarian expression and gonadotropin regulation was hitherto unknown. The induction of three of these genes, encoding cutaneous fatty acid-binding protein, the interleukin-4 receptor alpha chain, and prepronociceptin, was confirmed and further characterized by Northern blot analysis. In addition, in situ hybridization analysis showed that hCG administration resulted in exclusive or predominant expression of all three genes in theca cells. These results demonstrate that DNA arrays can be used to identify genes regulated during the periovulatory period, thus contributing to a more detailed understanding of the molecular mechanisms of ovulation.

Expression of messenger ribonucleic acid for the antiapoptosis gene P11 in the rat ovary: gonadotropin stimulation in granulosa cells of preovulatory follicles.

P11, a member of the S100 family of calcium-binding proteins, has been shown to interact with BAD (Bcl-xL/Bcl-2-associated death promoter) in the yeast two-hybrid protein-protein interaction assay. Because overexpression of P11 dampens the proapoptotic activity of BAD in transfected cells, we tested the possibility that the expression of this antiapoptotic protein may be regulated by gonadotropins and other survival factors in the ovary. Northern blot analysis of ovaries obtained from prepubertal rats revealed an increased expression of P11 messenger RNA (mRNA) during prepubertal development in the theca cells of preantral and early antral follicles. Treatment of immature rats with PMSG did not affect P11 expression, whereas treatment of PMSG-primed rats with an ovulatory dose of human (h)CG stimulated ovarian P11 mRNA within 6-9 h in the granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time-dependent stimulation of P11 by gonadotropins. In addition, treatment of cultured preovulatory follicles with MDL-12,330A, an inhibitor of adenylate cyclase, inhibited LH-stimulated P11 mRNA, whereas treatment with forskolin, an adenylate cyclase activator, but not the protein kinase C activator, 2-O-tetradecanol-phorbal-13-acetate, mimicked the LH action, suggesting the role of adenylate cyclase activation in P11 expression. Treatment with other follicle survival factors, including the epidermal growth factor, the basic fibroblast growth factor, and interleukin-1beta, could also stimulate P11 expression in cultured preovulatory follicles. These results demonstrate the expression of P11 mRNA in theca cells of different-sized follicles and in granulosa cells of preovulatory follicles following gonadotropin stimulation, and suggest that P11 may mediate, at least partially, the survival action of gonadotropins during the ovulatory process.

Evolution and classification of cystine knot-containing hormones and related extracellular signaling molecules.

The cystine knot three-dimensional structure is found in many extracellular molecules and is conserved among divergent species. The identification of proteins with a cystine knot structure is difficult by commonly used pairwise alignments because the sequence homology among these proteins is low. Taking advantage of complete genome sequences in diverse organisms, we used a complementary approach of pattern searches and pairwise alignments to screen the predicted protein sequences of five model species (human, fly, worm, slime mold, and yeast) and retrieved proteins with low sequence homology but containing a typical cystine knot signature. Sequence comparison between proteins known to have a cystine knot three-dimensional structure (transforming growth factor-beta, glycoprotein hormone, and platelet-derived growth factor subfamily members) identified new crucial amino acid residues (two hydrophilic amino acid residues flanking cysteine 5 of the cystine knot). In addition to the well known members of the cystine knot superfamily, novel subfamilies of proteins (mucins, norrie disease protein, von Willebrand factor, bone morphogenetic protein antagonists, and slit-like proteins) were identified as putative cystine knot-containing proteins. Phylogenetic analysis revealed the ancient evolution of these proteins and the relationship between hormones [e.g. transforming growth factor-beta (TGFbeta)] and extracellular matrix proteins (e.g. mucins). They are absent in the unicellular yeast genome but present in nematode, fly, and higher species, indicating that the cystine knot structure evolved in extracellular signaling molecules of multicellular organisms. All data retrieved by this study can be viewed at http://hormone.stanford.edu/.

Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor.

Adaptive stress responses mediated by the endocrine, autonomic, cardiovascular and immune systems are essential for the survival of the individual. Initial stress-induced responses provide a vital short-term metabolic lift, but prolonged or inappropriate exposure to stress can compromise homeostasis thereby leading to disease. This 'fight-or-flight' response is characterized by the activation of the corticotropin-releasing hormone (CRH)-adrenocorticotropin-glucocorticoid axis, mediated by the type 1 CRH receptor. In contrast, the type 2 CRH receptor mediates the stress-coping responses during the recovery phase of stress. We identified human stresscopin (SCP) and stresscopin-related peptide (SRP) as specific ligands for the type 2 CRH receptor. The genes encoding these peptides were expressed in diverse peripheral tissues as well as in the central nervous system. Treatment with SCP or SRP suppressed food intake, delayed gastric emptying and decreased heat-induced edema. Thus SCP and SRP might represent endogenous ligands for maintaining homeostasis after stress, and could allow the design of drugs to ameliorate stress-related diseases.

Mutations in the follicle-stimulating hormone receptor and familial dizygotic twinning.

Concentrations of follicle-stimulating hormone (FSH) have an important role in multiple ovulation. An association has been reported between mutations in the FSH receptor (FSHR) in a family with increased twinning frequency. We sequenced the transmembrane region of FSHR (located on chromosome 2) in 21 unrelated mothers of dizygotic twins and found no differences to the published sequence. A linkage study of 183 sister pairs and trios, in which all sisters had given birth to spontaneous dizygotic twins, excluded linkage to this region of chromosome 2. We conclude that mutations in FSHR are not a common cause of familial dizygotic twinning.

Ovarian gene database.

OBJECTIVE: The entire human genome will be sequenced in September 2000. Facing the exponential increase of data in the GenBank at the National Center for Biotechnology Information, reproductive biologists are being bombarded with massive amounts of information on diverse genes. It is becoming increasingly difficult for individual investigators to sort out the diverse genetic and physiologic information on the localization and function of different genes in the ovary. To alleviate the present situation, we have taken advantage of the accessibility of the Internet and initiated a project that serves the entire ovarian research community. RESULTS AND DISCUSSION: The Ovarian Kaleidoscope database provides information regarding biologic function, expression pattern, and regulation of genes that are expressed in the ovary. In addition, it serves as a gateway to other online information resources relevant to ovarian research by offering results from original papers and data about nucleotide and amino acid sequences, and human and murine mutation phenotypes. All references are linked by hypertext to PubMed and additional links to sequence databases are also included. This information is accessible online and searchable not only by gene name but also by criteria such as the cellular and ovarian function of the gene product, the expression of genes in different ovarian cell types, or their association with specific ovarian phenotypes.

Müllerian inhibitory substance induces growth of rat preantral ovarian follicles.

Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is best known as the hormone that regulates the regression of the Müllerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Müllerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis.

Characterization of two fly LGR (leucine-rich repeat-containing, G protein-coupled receptor) proteins homologous to vertebrate glycoprotein hormone receptors: constitutive activation of wild-type fly LGR1 but not LGR2 in transfected mammalian cells.

The receptors for lutropin (LH), FSH, and TSH belong to the large G protein-coupled receptor (GPCR) superfamily and are unique in having a large N-terminal extracellular (ecto-) domain important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of a large family of the leucine-rich repeat-containing, G protein-coupled receptors (LGRs) with at least seven members in mammals. Based on the sequences of mammalian glycoprotein hormone receptors, we have identified a new LGR in Drosophila melanogaster and named it as fly LGR2 to distinguish it from the previously reported fly LH/FSH/TSH receptor (renamed as fly LGR1). Genomic analysis indicated the presence of 10 exons in fly LGR2 as compared with 16 exons in fly LGR1. The deduced fly LGR2 complementary DNA (cDNA) showed 43 and 64% similarity to the fly LGR1 in the ectodomain and transmembrane region, respectively. Comparison of 12 LGRs from diverse species indicated that these proteins can be divided into three subfamilies and fly LGR1 and LGR2 belong to different subfamilies. Potential signaling mechanisms were tested in human 293T cells overexpressing the fly receptors. Of interest, fly LGR1, but not LGR2, showed constitutive activity as reflected by elevated basal cAMP production in transfected cells. The basal activity of fly LGR1 was further augmented following point mutations of key residues in the intracellular loop 3 or transmembrane VI, similar to those found in patients with familial male precocious puberty. The present study reports the cloning of fly LGR2 and indicates that the G protein-coupling mechanism is conserved in fly LGR1 as compared with the mammalian glycoprotein hormone receptors. The characterization of fly receptors with features similar to mammalian glycoprotein hormone receptors allows a better understanding of the evolution of this unique group of GPCRs and future elucidation of their ligand signaling mechanisms.

In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats.

Growth differentiation factor (GDF)-9 is a cystine knot-containing hormone of the transforming growth factor-beta superfamily produced by the oocyte. In GDF-9 null mice, follicle development is arrested at the primary stage and GDF-9 treatment in vitro enhances preantral follicle growth. Immature female rats were treated with recombinant GDF-9 for 7 or 10 days. At 10 days, treatment with GDF-9 augmented ovarian weights, concomitant with an increase in the number of primary and small preantral follicles by 30 and 60%, respectively. Furthermore, the number of primordial follicles was decreased by 29%, but the number of large preantral follicles was not affected. In contrast, treatment with FSH increased the number of small and large preantral follicles by 36 and 177% but did not influence the number of primary and primordial follicles. Immunoblot analysis showed an increase of CYP17, a theca cell marker, in the ovarian homogenate after treatment with GDF-9 but not FSH. The present results indicate that in vivo treatment with GDF-9 enhances the progression of primordial and primary follicles into small preantral follicles. Thus, GDF-9 treatment could provide an alternative approach to stimulate early follicle development in addition to the widely used FSH that acts mainly on the development of more advanced follicles.

Growth differentiation factor-9 stimulates rat theca-interstitial cell androgen biosynthesis.

Growth differentiation factor-9 (GDF-9) was shown recently to be essential for early follicular development, including the appearance of the theca layer. Theca cells provide the androgen substrate for aromatization and estrogen production by granulosa cells. Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, we have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and differentiation.

Dizygotic twinning is not linked to variation at the alpha-inhibin locus on human chromosome 2.

Natural multiple pregnancy in women leading to dizygotic (DZ) twins is familial and varies across racial groups, suggesting a genetic predisposition. Mothers of DZ twins have a higher incidence of spontaneous multiple ovulation and elevated FSH concentrations. FSH release is controlled by feedback of inhibin peptides from the ovary, and immunization against inhibin alpha-subunit results in an increased ovulation rate in animals. The inhibin alpha-subunit is therefore a candidate gene for mutations that may increase the frequency of DZ twinning. Restriction digests of a PCR product from exon 1 with the enzyme SpeI detects a C/T polymorphism at bp 128 with two alleles of 447 and 323/124 bp. The polymorphism was typed in 1,125 individuals from 326 pedigrees with 717 mothers of spontaneous DZ twins. The alpha-inhibin locus mapped within 3 centimorgans of D2S164, and linkage with DZ twinning was excluded [decimal log odds ratio (LOD) score, -2.81 at theta = 0]. There was complete exclusion of linkage (LOD, less than -2) of a gene conferring relative risk 1.8 (lambdas, >1.8) across the chromosome, except at the p-terminus region and a small peak (maximum LOD score, 0.6) in the region of D2S151-D2S326. Analysis using either recessive or dominant models excluded linkage with DZ twinning in this population (LOD score, less than -2.5) across chromosome 2. We conclude that dizygotic twinning is not linked to variation in the alpha-inhibin locus. The results also suggest that mutations in other candidates on chromosome 2, including the receptor for FSH and the betaB-inhibin subunit (INHBB) cannot be major contributors to risk for DZ twinning.

Hormonal regulation of early follicle development in the rat ovary.

Although earlier studies focused on the hormonal regulation of antral and preovulatory follicles, recent studies indicate the importance of the hormonal control mechanism for preantral follicles. The endocrine hormone FSH is not only a survival factor for early antral follicles but also a potent growth and differentiation factor for preantral follicles. In addition, KGF secreted by theca cells and c-kit ligand secreted by granulosa cells play paracrine roles in the regulation of preantral follicle growth and development. Furthermore oocyte-derived GDF-9 promotes the growth and differentiation of early follicles by acting on somatic cells in the follicle. It is likely that the genetic makeup of an oocyte could determine the secretion of oocyte hormones which would, in turn, regulate the growth and differentiation of the surrounding somatic cells of that follicle. A better understanding of the hormonal mechanisms underlying early follicle development could provide a refined culture system for the in vitro maturation of fertilizable oocytes and future design of fertility and contraceptive agents.