PubMed Health⌕ Search

Biomedical subjects

M M Matzuk

Publications and source records attributed to M M Matzuk.

At least 37 records · Page 2Linked to original sources
PubMed2000

Biochemical interactions of the neuronal pentraxins. Neuronal pentraxin (NP) receptor binds to taipoxin and taipoxin-associated calcium-binding protein 49 via NP1 and NP2.

Neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and neuronal pentraxin receptor (NPR) are members of a new family of proteins identified through interaction with a presynaptic snake venom toxin taipoxin. We have proposed that these three neuronal pentraxins represent a novel neuronal uptake pathway that may function during synapse formation and remodeling. We have investigated the mutual interactions of these proteins by characterizing their enrichment on taipoxin affinity columns; by expressing NP1, NP2, and NPR singly and together in Chinese hamster ovary cells; and by generating mice that fail to express NP1. NP1 and NP2 are secreted, exist as higher order multimers (probably pentamers), and interact with taipoxin and taipoxin-associated calcium-binding protein 49 (TCBP49). NPR is expressed on the cell membrane and does not bind taipoxin or TCBP49 by itself, but it can form heteropentamers with NP1 and NP2 that can be released from cell membranes. This is the first demonstration of heteromultimerization of pentraxins and release of a pentraxin complex by proteolysis. These processes are likely to directly effect the localization and function of neuronal pentraxins in neuronal uptake or synapse formation and remodeling.

Animals↗
PubMed2000

Revelations of ovarian follicle biology from gene knockout mice.

Autocrine, paracrine and endocrine factors are necessary for normal ovarian folliculogenesis. A number of studies have demonstrated that the pituitary hormones follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as granulosa cell-derived growth factors such as activins, inhibins, and kit ligand are necessary for follicle development. Recent knockout studies from our laboratory have demonstrated that mice lacking the pituitary proteins FSHbeta and activin receptor type II are infertile due to blocks at the pre-antral and antral follicle stages, respectively. Although the somatic cells of the ovary (the granulosa and theca cells) have long been implicated in ovarian function, only recently have we shown that the oocyte plays an essential role in controlling its own fate by influencing somatic cell functions. Mice lacking the oocyte-secreted growth factor, growth differentiation factor-9 (GDF-9), are infertile due to a block at the one-layer primary follicle stage, leading to secondary defects in thecal cell layer formation, oocyte growth and meiotic competence, and granulosa cell differentiation. Furthermore, using recombinant GDF-9, we demonstrate that GDF-9 also regulates cumulus expansion and expression of several key granulosa cell-specific genes. Thus, GDF-9 functions as an oocyte-secreted growth and differentiation factor during early and late folliculogenesis and at ovulation to regulate several key somatic cell functions essential for female reproduction.

Activins↗
PubMed2000

Glutathione synthesis is essential for mouse development but not for cell growth in culture.

Glutathione (GSH) is a major source of reducing equivalents in mammalian cells. To examine the role of GSH synthesis in development and cell growth, we generated mice deficient in GSH by a targeted disruption of the heavy subunit of gamma-glutamylcysteine synthetase (gammaGCS-HS(tm1)), an essential enzyme in GSH synthesis. Embryos homozygous for gammaGCS-HS(tm1) fail to gastrulate, do not form mesoderm, develop distal apoptosis, and die before day 8.5. Lethality results from apoptotic cell death rather than reduced cell proliferation. We also isolated cell lines from homozygous mutant blastocysts in medium containing GSH. These cells also grow indefinitely in GSH-free medium supplemented with N-acetylcysteine and have undetectable levels of GSH; further, they show no changes in mitochondrial morphology as judged by electron microscopy. These data demonstrate that GSH is required for mammalian development but dispensable in cell culture and that the functions of GSH, not GSH itself, are essential for cell growth.

Acetylcysteine↗
PubMed2000

Repeat instability and motor incoordination in mice with a targeted expanded CAG repeat in the Sca1 locus.

To elucidate the pathophysiology of spinocerebellar ataxia type 1 (SCA1) and to evaluate repeat length instability in the context of the mouse Sca1 gene, we generated knock-in mice by inserting an expanded tract of 78 CAG repeats into the mouse Sca1 locus. Mice heterozygous for the CAG expansion show intergenerational repeat instability (+2 to -6) at a much higher frequency in maternal transmission than in paternal transmission. The majority of changes transmitted through the female germline were small contractions, as in humans, whereas small expansions occurred more frequently in paternal transmission. The frequency of intergenerational changes was age dependent for both paternal and maternal transmissions. Mice homozygous for mutant ataxin-1 on a C57BL/6J-129/SvEv mixed background performed significantly less well on the rotating rod than did wild-type littermates at 9 months of age, although they were not ataxic by cage behavior. Histological examination of brain tissue from mutant mice up to 18 months of age revealed none of the neuropathological changes observed in other transgenic models overexpressing expanded polyglutamine tracts. These data suggest that, even with 78 glutamines, prolonged exposure to mutant ataxin-1 at endogenous levels is necessary to produce a neurological phenotype reminiscent of human SCA1. Pathogenesis is thus a function of polyglutamine length, protein levels and duration of neuronal exposure to the mutant protein.

Amino Acid Sequence↗
PubMed2000

Smad5 is essential for left-right asymmetry in mice.

Left-right (L-R) asymmetry of the vertebrate body plan is established from an originally morphologically symmetric embryo. Recent studies have implicated several TGF-beta family signaling proteins (i.e., nodal, lefty-1, lefty-2, activin receptor type IIB, and Smad2) in L-R axis determination in the mouse. However, the genetic pathways underlying L-R patterning are still unclear. Smad5 is a downstream component in the TGF-beta family signaling cascade, and lack of Smad5 results in embryonic lethality between E9.5 and E11.5. In this report, we demonstrate that Smad5 mutant embryos have defects in heart looping and embryonic turning which are the first signs of L-R asymmetry in mice. To gain more insights into the molecular basis of the laterality defects in the Smad5-deficient embryos, we examined the expression of lefty-1, lefty-2, nodal, and Pitx2 since the asymmetric expression of these genes always closely correlates with the direction of heart looping and embryonic turning. In the absence of Smad5, lefty-1 was expressed at very low or undetectable levels, while nodal, lefty-2, and Pitx2 were expressed bilaterally. These data suggest that Smad5 is upstream of lefty-1, nodal, and lefty-2, and as a consequence also of Pitx2, and Smad5 is essential for L-R axis determination.

Animals↗
PubMed2000

Oocyte-expressed TGF-beta superfamily members in female fertility.

Folliculogenesis is regulated by the interplay of extraovarian and intraovarian factors, and the importance of each type of regulation varies depending on the developmental stage of the follicle. Preantral follicle development is regulated predominantly by factors produced locally within the ovary and within the follicle itself. The oocyte has been shown to produce soluble factor(s), which regulate a number of processes in follicular development, including cumulus expansion in the periovulatory period. Members of the TGFbeta superfamily are potent regulators of cell proliferation and differentiation in a number of organ systems, and three members, growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15) and BMP-6 are expressed by the oocyte and may mediate effects attributed to the oocyte. Based on knockout mouse models BMP-6 does not play an essential role in ovarian function, but GDF-9 is absolutely required for preantral follicle development. GDF-9 also alters the periovulatory expression of granulosa cell genes and stimulates cumulus expansion. Although BMP-15 is expressed identically to GDF-9, its role in regulating ovarian function is still unknown. This review examines the similarities and differences in sequence, expression, and function of the oocyte-expressed TGFbeta family members with respect to regulating folliculogenesis.

Animals↗
PubMed2000

Infant vocalization, adult aggression, and fear behavior of an oxytocin null mutant mouse.

Previous studies have shown that oxytocin (OT)-deficient female mice produced by homologous recombination fail to lactate but exhibit normal parturition and reproductive behaviors. We examined the ultrasonic vocalizations of infant mice and the subsequent aggressive and fear behavior of adult male OT knockout (OT-KO) mice. Infant OT-KO mice were less vocal than wild-type (WT) control mice during separations from the mother and peers. Adult OT-KO males were generally more aggressive in isolation-induced and resident-intruder tests of aggression and less fearful in the plus maze and acoustic startle reflex tests than WT controls. Although the increase in tests of aggression was robust for OT-KO males from obligate litters (progeny of homozygous x homozygous crossings), the increase in aggression was reduced during tests for OT-KO males derived from nonobligate mating (progeny of heterozygous x heterozygous crossings), suggesting that the OT-KO genotype was not, by itself, responsible for the changes in adult behavior. We conclude that the absence of exposure to OT during development was associated with abnormalities in the development of emotional behavior.

Age Factors↗
PubMed2000

Activin A promotes progenitor differentiation into photoreceptors in rodent retina.

Activins are TGF beta-like proteins that were first discovered for their actions on the reproductive system, but have subsequently been shown to play a role in a variety of developmental processes. Previous studies have demonstrated that activins and their receptors are present in the developing retina, as well as other regions of the embryonic nervous system. We used both in vitro and in vivo approaches to test for functions of activin during retinal development. We found that activin A treatment of embryonic day 18 rat retinal cultures causes the progenitor cells in the cultures to exit the cell cycle and differentiate into rod photoreceptors. This effect is dose-dependent and the promotion of rod photoreceptor differentiation is specific, since the other primary retinal neurons generated in these cultures, the C1+ amacrine cells, are not affected by activin A treatment. Mice with homozygous deletion of the activin betaA gene show a specific decrease in the number of rod photoreceptors compared to wild-type or heterozygous littermates. These data demonstrate that activin A is an important regulator of photoreceptor differentiation in the developing retina.

Activins↗
PubMed2000

Social amnesia in mice lacking the oxytocin gene.

The development of social familiarity in rodents depends predominantly on olfactory cues and can critically influence reproductive success. Researchers have operationally defined this memory by a reliable decrease in olfactory investigation in repeated or prolonged encounters with a conspecific. Brain oxytocin (OT) and vasopressin (AVP) seem to modulate a range of social behaviour from parental care to mate guarding. Pharmacological studies indicate that AVP administration may enhance social memory, whereas OT administration may either inhibit or facilitate social memory depending on dose, route or paradigm. We found that male mice mutant for the oxytocin gene (Oxt-/-) failed to develop social memory, whereas wild-type (Oxt+/+) mice showed intact social memory. Measurement of both olfactory foraging and olfactory habituation tasks indicated that olfactory detection of non-social stimuli is intact in Oxt-/- mice. Spatial memory and behavioural inhibition measured in a Morris water-maze, Y-maze, or habituation of an acoustic startle also seemed intact. Treatment with OT but not AVP rescued social memory in Oxt-/- mice, and treatment with an OT antagonist produced a social amnesia-like effect in Oxt+/+ mice. Our data indicate that OT is necessary for the normal development of social memory in mice and support the hypothesis that social memory has a neural basis distinct from other forms of memory.

Amnesia, Retrograde↗
PubMed2000

Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions.

The activins (dimers of betaA or betaB subunits, encoded by the genes Inhba and Inhbb, respectively) are TGF-beta superfamily members that have roles in reproduction and development. Whereas mice homozygous for the Inhba-null allele demonstrate disruption of whisker, palate and tooth development, leading to neonatal lethality, homozygous Inhbb-null mice are viable, fertile and have eye defects. To determine if these phenotypes were due to spatiotemporal expression differences of the ligands or disruption of specific ligand-receptor interactions, we replaced the region of Inhba encoding the mature protein with Inhbb, creating the allele Inhbatm2Zuk (hereafter designated InhbaBK). Although the craniofacial phenotypes of the Inhba-null mutation were rescued by the InhbaBK allele, somatic, testicular, genital and hair growth were grossly affected and influenced by the dosage and bioactivity of the allele. Thus, functional compensation within the TGF-beta superfamily can occur if the replacement gene is expressed appropriately. The novel phenotypes in these mice further illustrate the usefulness of insertion strategies for defining protein function.

Activins↗
PubMed2000

Activin betaC and betaE genes are not essential for mouse liver growth, differentiation, and regeneration.

The liver is an essential organ that produces several serum proteins, stores vital nutrients, and detoxifies many carcinogenic and xenobiotic compounds. Various growth factors positively regulate liver growth, but only a few negative regulators are known. Among the latter are the transforming growth factor beta (TGF-beta) superfamily members TGF-beta1 and activin A. To study the function of novel activin family members, we have cloned and generated mice deficient in the activin betaC and betaE genes. Expression analyses demonstrated that these novel genes are liver specific in adult mice. Here, we show by RNase protection that activin betaC transcripts are present in the liver beginning at embryonic day 11.5 (E11.5) whereas activin betaE expression is detected starting from E17.5. Gene targeting in embryonic stem cells was used to generate mice with null mutations in either the individual activin betaC and betaE genes or both genes. In contrast to the structurally related activin betaA and betaB subunits, which are necessary for embryonic development and pituitary follicle-stimulating hormone homeostasis, mice deficient in activin betaC and betaE were viable, survived to adulthood, and demonstrated no reproductive abnormalities. Although activin betaC and betaE mRNAs are abundantly expressed in the liver of wild-type mice, the single and double mutants did not show any defects in liver development and function. Furthermore, in the homozygous mutant mice, liver regeneration after >70% partial hepatectomy was comparable to that in wild-type mice. Our results suggest that activin betaC and betaE are not essential for either embryonic development or liver function.

Activins↗
PubMed2000

Reproductive defects in gamma-glutamyl transpeptidase-deficient mice.

Mice deficient in gamma-glutamyl transpeptidase (GGT) are growth retarded as a result of cysteine deficiency secondary to excessive glutathione excretion in urine and display coat color defects and cataracts. Although GGT is widely expressed throughout the mouse reproductive axis, little is known about its role in reproduction. Here, we present an analysis of the reproductive phenotypes of GGT-deficient mice. Mutant male mice have reduced testis and seminal vesicle size and suppressed serum insulin-like growth factor I and FSH levels and are infertile. Although these mice are severely oligospermic, histological analysis of testes reveals grossly normal stages of spermatogenesis, including late stage spermatids, but the tubule diameter is reduced. GGT-deficient female mice are also hypogonadal and infertile. At 6 weeks of age, the ovaries of mutant mice are histologically indistinguishable from those of its wild-type counterpart. However, the absence of antral follicles and corpora lutea and follicular degeneration are apparent by 11-13 weeks. In addition, immature female mutant mice (at 21-23 days) are insensitive to exogenous gonadotropin administration and fail to superovulate, suggesting an intraovarian defect. Consistent with these mutant phenotypes, HPLC analysis of adult mutant testes and ovaries showed a reduction in intracellular cysteine levels. Administration of N-acetylcysteine in the drinking water beginning on day 21 to mutant mice for 2 weeks restored testis, seminal vesicle, and ovary sizes to values comparable to those in wild-type mice. Furthermore, N-acetylcysteine-fed (continuously) mutant male and female mice were fertile and produced normal numbers of offspring when mated to wild-type control mice. These results demonstrate that GGT itself is not necessary for reproductive function. However, GGT plays an important role in cysteine homeostasis within the mouse reproductive axis.

Acetylcysteine↗
PubMed2000

Follistatin is a modulator of gonadal tumor progression and the activin-induced wasting syndrome in inhibin-deficient mice.

Inhibins and activins are dimeric proteins belonging to the transforming growth factor-beta superfamily. Follistatin is an activin-binding protein that antagonizes the function of activin via binding to its beta-subunits. Previously, we demonstrated that mice deficient in inhibin develop ovarian and testicular sex cord-stromal tumors of granulosa and Sertoli cell origin, with 100% penetrance as early as 4 weeks of age. Overproduction of activins in the serum directly causes a cachexia-like wasting syndrome that results in lethality of these mice at an early stage after the onset of the tumors. In an independent set of studies, overexpression of mouse follistatin using the mouse metallothionein I promoter in transgenic mice led to gonadal defects and eventual infertility, primarily due to local effects of follistatin in these tissues. Activin has a positive growth effect on gonadal tumor cells in culture and directly causes the cancer cachexia-like syndrome in inhibin-deficient mice via interaction with activin receptor type IIA in livers and stomachs. We therefore hypothesized that an activin antagonist such as follistatin can act as a physiological modifier, either locally or via the serum, to block the activin-mediated cancer cachexia-like syndrome in inhibin-deficient mice and/or slow the progression of gonadal cancers in these mice. To test this hypothesis, we generated mice that are homozygous mutant for the inhibin alpha null allele (i.e. inham1/inham1) and carry the mouse metallothionein I follistatin (MT-FS) transgene. Our results show that gonadal tumors that are histologically similar in most, but not all, cases to the tumors in inhibin-deficient mice develop in these inham1/inham1, MT-FS+ mice. However, inham1/inham1, MT-FS+ mice exhibit a less severe wasting syndrome, lower serum activin levels, and a statistically significant prolonged survival in a number of cases compared with mice deficient in inhibin alone. Thus, follistatin can act as a modulator of tumor growth and the activin-induced cancer cachexia-like syndrome in inhibin-deficient mice.

Activins↗
PubMed2000

Regulable expression of inhibin A in wild-type and inhibin alpha null mice.

Exogenous regulation of protein expression creates the potential to examine the consequences of homeostatic Dysregulation in many physiological systems and, when used in transgenic mice, provides the capability of restoring a gene product to its knockout background without antigenicity issues. In this study, we used a mifeprisone-inducible system (the GeneSwitch system) to regulate the expression of inhibin A from the liver of mice. Inhibin is a heterodimeric protein (alpha/beta) wherein one of its subunits (beta) is capable of homodimerizing to form its physiological antagonist, activin (beta/beta). Inhibin is also expressed in two forms, A and B, as determined by the subtype of beta-subunit that dimerizes with the alpha-subunit (alpha/betaA or alpha/betaB). To utilize the GeneSwitch system, transgenic transactivator mice with liver-specific expression of a mifepristone-activated chimeric nuclear receptor (GLVP) were crossed with transgenic target mice containing a GVLP-responsive promoter upstream of polio-virus IRES (internal ribosome entry site)-linked sequences coding for the alpha- and beta-subunits of inhibin A. This intercross produced "bigenic" mice capable of regulable expression of inhibin A from the liver. Overexpression of inhibin A in wild-type mice produced a phenotype wherein males had decreased testis size and females had a block in folliculogenesis at the early antral stage, findings similar to activin type IIA receptor (ActRIIA) null mice. These phenotypes were most likely due to suppressed serum FSH, confirming that the liver-derived inhibin A was secreted into the serum to down-regulate pituitary FSH levels. Furthermore, the generation of bigenic mice in the inhibin alpha null background allowed for the induction of inhibin A in inhibin alpha null male mice with subsequent rescue of these mice from their gonadal tumor-induced lethal phenotype. This work demonstrates the in vivo production of a heterodimeric hormone from a single inducible promoter to study its therapeutic and physiological effects. In addition, these studies are the first example of an inducible system being used to prevent a lethal knockout phenotype in an animal model.

Animals↗
PubMed1999

Transgenic models to study gonadotropin function: the role of follicle-stimulating hormone in gonadal growth and tumorigenesis.

The role of FSH in gonadal tumorigenesis and, in particular, in human ovarian cancer has been debated. It is also unclear what role the elevated FSH levels in the inhibin-deficient mouse play in the gonadal tumorigenesis. To directly assess the role of FSH in gonadal growth, differentiation, and gonadal tumorigenesis, we have generated both gain-of-function and loss-of-function transgenic mutant mice. In the gain-of-function model, we have generated transgenic mice that ectopically overexpress human FSH from multiple tissues using a mouse metallothionein-1 promoter, achieving levels far exceeding those seen in postmenopausal women. Male transgenic mice are infertile despite normal testicular development and demonstrate enlarged seminal vesicles secondary to elevated serum testosterone levels. Female transgenic mice develop highly hemorrhagic and cystic ovaries, have elevated serum estradiol and progesterone levels, and are infertile, mimicking the features of human ovarian hyperstimulation and polycystic ovarian syndromes. Furthermore, the female transgenic mice develop enlarged and cystic kidneys and die between 6-13 weeks as a result of urinary bladder obstruction. In a complementary loss-of-function approach, we have generated double-homozygous mutant mice that lack both inhibin and FSH by a genetic intercross. In contrast to male mice lacking inhibin alone, 95% of which die of a cancer cachexia-like syndrome by 12 weeks of age, only 30% of the double-mutant male mice lacking both FSH and inhibin die by 1 yr of age. The remaining double-mutant male mice develop slow-growing and less hemorrhagic testicular tumors, which are noted after 12 weeks of age, and have minimal cachexia. Similarly, the double-mutant female mice develop slow-growing, less hemorrhagic ovarian tumors, and 70% of these mice live beyond 17 weeks. The double-mutant mice demonstrate minimal cachexia in contrast to female mice lacking only inhibin, which develop highly hemorrhagic ovarian tumors, leading to cachexia and death by 17 weeks of age in 95% of the cases. The milder cachexia-like symptoms of the inhibin and FSH double-mutant mice are correlated with low levels of serum estradiol and activin A and reduced levels of aromatase mRNA in the gonadal tumors. Based on these and our previous genetic analyses, we conclude that elevated FSH levels do not directly cause gonadal tumors. However, these results suggest FSH is an important trophic modifier factor for gonadal tumorigenesis in inhibin-deficient mice.

Activins↗
PubMed1999

Molecular characterization of the follicle defects in the growth differentiation factor 9-deficient ovary.

Growth differentiation factor-9 (GDF-9), a secreted member of the transforming growth factor-beta superfamily, is expressed at high levels in the mammalian oocyte beginning at the type 3a primary follicle stage. We have previously demonstrated that GDF-9-deficient female mice are infertile because of an early block in folliculogenesis at the type 3b primary follicle stage. To address the molecular defects that result from the absence of GDF-9, we have analyzed the expression of several important ovarian marker genes. The major findings of our studies are as follows: 1) There are no detectable signals around GDF-9-deficient follicles for several theca cell layer markers [i.e. 17alpha-hydroxylase, LH receptor (LHR), and c-kit, the receptor for kit ligand]. This demonstrates that in the absence of GDF-9, the follicles are incompetent to emit a signal that recruits theca cell precursors to surround the follicle; 2) The primary follicles of GDF-9-deficient mice demonstrate an up-regulation of kit ligand and inhibin-alpha. This suggests that these two important secreted growth factors, expressed in the granulosa cells, may be directly regulated in a paracrine fashion by GDF-9. Up-regulation of kit ligand, via signaling through c-kit on the oocyte, may be directly involved in the increased size of GDF-9-deficient oocytes and the eventual demise of the oocyte; 3) After loss of the oocyte, the cells of the GDF-9-deficient follicles remain in a steroidogenic cluster that histologically resembles small corpora lutea. However, at the molecular level, these cells are positive for both luteal markers (e.g. LHR and P-450 side chain cleavage) and nonluteal markers (e.g. inhibin alpha and P-450 aromatase). This demonstrates that initially the presence of the oocyte prevents the expression of luteinized markers, but that the absence of GDF-9 at an early timepoint alters the differentiation program of the granulosa cells; and 4) As demonstrated by staining with either proliferating cell nuclear antigen (PCNA) or Ki-67 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) labeling, the granulosa cells of GDF-9-deficient type 3b primary follicles fail to proliferate but also fail to undergo cell death. This suggests that granulosa cells of type 3b follicles require GDF-9 for continued growth and also to become competent to undergo apoptosis, possibly through a differentiation event Thus, these studies have enlightened us as to the paracrine roles of GDF-9 as well as the normal steps of granulosa cell and theca cell growth and differentiation within ovarian follicles.

Activins↗
PubMed1999

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.

Animals↗