Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene

The formation of estrogens from C19 steroids is catalyzed by aromatase cytochrome P450 (P450arom), the product of the cyp19 gene. The actions of estrogen include dimorphic anatomical, functional, and behavioral effects on the development of both males and females, considerations that prompted us to examine the consequences of deficiency of aromatase activity in mice. Mice lacking a functional aromatase enzyme (ArKO) were generated by targeted disruption of the cyp19 gene. Male and female ArKO mice were born with the expected Mendelian frequency from F1 parents and grew to adulthood. Female ArKO mice at 9 weeks of age displayed underdeveloped external genitalia and uteri. Ovaries contained numerous follicles with abundant granulosa cells and evidence of antrum formation that appeared arrested before ovulation. No corpora lutea were present. Additionally the stroma were hyperplastic with structures that appeared to be atretic follicles. Development of the mammary glands approximated that of a prepubertal female. Examination of male ArKO mice of the same age revealed essentially normal internal anatomy but with enlargement of the male accessory sex glands because of increased content of secreted material. The testes appeared normal. Male ArKO mice are capable of breeding and produce litters of approximately average size. Whereas serum estradiol levels were at the limit of detection, testosterone levels were elevated, as were the levels of follicle-stimulating hormone and luteinizing hormone. The phenotype of these animals differs markedly from that of the previously reported ERKO mice, in which the estrogen receptor α is deleted by targeted disruption.

Impairing follicle-stimulating hormone (FSH) signaling in vivo: Targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R −/− mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R −/− mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Mérino ewes

Ewes from the Booroola strain of Australian Mérino sheep are characterized by high ovulation rate and litter size. This phenotype is due to the action of the FecBB allele of a major gene named FecB, as determined by statistical analysis of phenotypic data. By genetic analysis of 31 informative half-sib families from heterozygous sires, we showed that the FecB locus is situated in the region of ovine chromosome 6 corresponding to the human chromosome 4q22–23 that contains the bone morphogenetic protein receptor IB (BMPR-IB) gene encoding a member of the transforming growth factor-β (TGF-β) receptor family. A nonconservative substitution (Q249R) in the BMPR-IB coding sequence was found to be associated fully with the hyperprolificacy phenotype of Booroola ewes. In vitro, ovarian granulosa cells from FecBB/FecBB ewes were less responsive than granulosa cells from FecB+/FecB+ ewes to the inhibitory effect on steroidogenesis of GDF-5 and BMP-4, natural ligands of BMPR-IB. It is suggested that in FecBB/FecBB ewes, BMPR-IB would be inactivated partially, leading to an advanced differentiation of granulosa cells and an advanced maturation of ovulatory follicles.

The type I BMP receptor BmprIB is essential for female reproductive function

Maintenance of female reproductive competence depends on the actions of several hormones and signaling factors. Recent reports suggest roles for bone morphogenetic proteins (BMPs) in early stages of folliculogenesis. A role for the type I BMP receptor BmprIB as a regulator of ovulation rates in sheep has been described recently, but little is known about the roles of BMP signaling pathways in other aspects of reproductive function. We report here that BMPRIB is essential for multiple aspects of female fertility. Mice deficient in BmprIB exhibit irregular estrous cycles and an impaired pseudopregnancy response. BmprIB mutants produce oocytes that can be fertilized in vitro, but defects in cumulus expansion prevent fertilization in vivo. This defect is associated with decreased levels of aromatase production in granulosa cells. Unexpectedly, levels of mRNA for cyclooxygenase 2, an enzyme required for cumulus expansion, are increased. BmprIB mutants also exhibit a failure in endometrial gland formation. The expression of BmprIB in uterine linings suggests that these defects are a direct consequence of loss of BMP signaling in this tissue. In summary, these studies demonstrate the importance of BMP signaling pathways for estrus cyclicity, estradiol biosynthesis, and cumulus cell expansion in vivo and reveal sites of action for BMP signaling pathways in reproductive tissues.

Remodeling of extracellular matrix at ovulation of the bovine ovarian follicle

Using immunohistochemistry and RNA analyses we examined the fate of components of a newly identified matrix that develops between granulosa cells (focimatrix, abbreviated from focal intraepithelial matrix) and of the follicular basal lamina in ovulating bovine ovarian follicles. Pre- and postovulatory follicles were generated by treatment with estradiol (Day 1), progesterone (Days 1-10), and prostaglandin analogue (Day 9) with either no further treatment (Group 1, n = 6) and or with 25 mg porcine LH (Day 11, Group 2, n = 8 or Day 10, Group 3, n = 8) and ovariectomy on Day 12 (12-14 hr post LH in Group 2, 38-40.5 hr in Group 3). In the time frame examined no loss of follicular basal lamina laminin chains beta 2 and gamma 1 or nidogen 1 was observed. In the follicular basal lamina collagen type IV alpha 1 and perlecan were present prior to ovulation; after ovulation collagen type IV alpha 1 was discontinuously distributed and perlecan was absent. Versican in the theca interna adjacent to the follicular basal lamina in preovulatory follicles was not observed post ovulation, however, the granulosa cells then showed strong cytoplasmic staining for versican. Expression of versican isoforms V0, V1, and V3 was detected at all stages. Focimatrix was observed in preovulatory follicles. It contained collagen type IV alpha 1, laminins beta 2 and gamma 1, nidogen 1 and perlecan and underwent changes in composition similar to that of the follicular basal lamina. In conclusion focimatrix and the follicular basal lamina are degraded at ovulation. Individual components are lost at different times.

Association of follicle stimulating hormone receptor promoter with ovarian response in IVF-ET patients

Background: Poor ovarian response phenomenon has been observed in some of the in vitro fertilization-embryo transfer patients. Some investigations found that follicle stimulating hormone receptor (FSHR) gene plays a role in the process, but no direct evidence shows the correlation between genotypes of FSHR and ovarian response. Objective: Exploring the molecular mechanism behind the mutation of FSHR promoter association with ovarian granulosa cells and poor ovarian response. Materials and Methods: This cross sectional study was performed using 158 women undergoing the controlled short program ovarian stimulation for IVF treatment. The 263 bp DNA fragments before the follicle stimulating hormone (FSH) receptor 5' initiation site were sequenced in the patients under IVF cycle, 70 of which had poor ovarian response and 88 showed normal ovarian responses. Results: With a mutation rate of 40%, 63 in 158 cases showed a 29th site G→A point mutation; among the mutated cases, the mutation rate of the poor ovarian responders was significantly higher than the normal group (60% versus 23.9%; χ2=21.450, p<0.001). Besides, the variability was also obvious in antral follicle count, and ovum pick-ups. The estradiol peak values and the number of mature eggs between the two groups had significant difference. However, there was no obvious variability (t=0.457, p=0.324) in the basic FSH values between the two groups (normal group, 7.2±2.3 U/L; mutation group, 7.1±2.0 U/L). Conclusion: The activity of FSHR promoter is significantly affected by the 29th site G→A mutation that will weaken promoter activity and result in poor response to FSH.

Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles

Ovarian follicle development is regulated through endocrine and local mechanisms. Increasing evidence indicates roles for transforming growth factor beta superfamily members, including inhibins and activins. We recently identified divergent expression of mRNAs encoding activin receptors (ActR) and inhibin co-receptor betaglycan in chicken follicles at different stages of maturation. Here, we compare the actions of LH and FSH (0, 1, 10, 100 ng/ml) on levels of mRNA for ActRI, ActRIIA, ActRIIB and betaglycan in chicken granulosa and theca cells (GC and TC) from preovulatory (F1) and prehierarchical (6-8 mm) follicles. The expression of mRNAs for LH-R and FSH-R and production of inhibin A, oestradiol and progesterone were also quantified. FSH decreased ActRIIB and ActRI mRNA levels in 6-8 mm GC, whereas LH increased the mRNA levels. Both LH and FSH enhanced ActRIIA (5- and 8.5-fold) and betaglycan mRNA expression (2- and 3.5-fold) in 6-8 mm GC. In 6-8 mm TC, LH and FSH both increased the betaglycan mRNA level (7- and 3.5-fold respectively) but did not affect ActRI, ActRIIA and ActRIIB transcript levels. In F1 GC, both LH and FSH stimulated ActRI (2- and 2.4-fold), ActRIIB (3.2- and 2.7-fold) and betaglycan (7- and 4-fold) mRNA levels, while ActRIIA mRNA was unaffected. In F1 TC, LH and FSH reduced ActRIIA (35-50%) and increased (4.5- and 7.6-fold) betaglycan mRNA, but had no effect on ActRI and ActRIIB transcript levels. Results support the hypothesis that expression of ActR and betaglycan are differentially regulated by gonadotrophins during follicle maturation in the hen. This may represent an important mechanism for fine-tuning follicle responsiveness to local and systemic activins and inhibins.

Transcription factor GATA4 and apoptotic TRAIL pathway in granulosa cell function and tumors

Normal growth and development require the precise control of gene expression. Transcription factors are proteins that regulate gene expression by binding specific sequences of DNA. Abnormalities in transcription are implicated in a variety of human diseases, including cancer, endocrine disorders and birth defects. Transcription factor GATA4 has emerged as an important regulator of normal development and function in a variety of endoderm- and mesoderm- derived tissues, including gut, heart and several endocrine organs, such as gonads. Mice harboring a null mutation of Gata4 gene die during embryogenesis due to failure in heart formation, complicating the study of functional role of GATA4 in other organs. However, the expression pattern of GATA4 suggests it may play a role in the regulation of ovarian granulosa cell development, function and apoptosis. This premise is supported by in vitro studies showing that GATA4 regulates several steroidogenic enzymes as well as auto-, para- and endocrine signaling molecules important for granulosa cell function. This study assessed the in vivo role of GATA4 for granulosa cell function by utilizing two genetically modified mouse strains. The findings in the GATA4 deficient mice included delayed puberty, impaired fertility and signs of diminished estrogen production. At the molecular level, the GATA4 deficiency leads to attenuated expression of central steroidogenic genes, Steroidogenic acute regulatory protein (StAR), Side-chain cleavage (SCC), and aromatase as a response to stimulations with exogenous gonadotropins. Taken together, these suggest GATA4 is necessary for the normal ovarian function and female fertility. Programmed cell death, apoptosis, is a crucial part of normal ovarian development and function. In addition, disturbances in apoptosis have been implicated to pathogenesis of human granulosa cell tumors (GCTs). Apoptosis is controlled by extrinsic and intrinsic pathways. The intrinsic pathway is regulated by members of Bcl-2 family, and its founding member, the anti-apoptotic Bcl-2, is known to be important for granulosa cell survival. This study showed that the expression levels of GATA4 and Bcl-2 correlate in the human GCTs and that GATA4 regulates Bcl-2 expression, presumably by directly binding to its promoter. In addition, disturbing GATA4 function was sufficient to induce apoptosis in cultured GCT- derived cell line. Taken together, these results suggest GATA4 functions as an anti-apoptotic factor in GCTs. The extrinsic apoptotic pathway is controlled by the members of tumor necrosis factor (TNF) superfamily. An interesting ligand of this family is TNF-related apoptosis-inducing ligand (TRAIL), possessing a unique ability to selectively induce apoptosis in malignant cells. This study characterized the previously unknown expression of TRAIL and its receptors in both developing and adult human ovary, as well as in malignant granulosa cell tumors. TRAIL pathway was shown to be active in GCTs suggesting it may be a useful tool in treating these malignancies. However, more studies are required to assess the function of TRAIL pathway in normal ovaries. In addition to its ability to induce apoptosis in GCTs, this study revealed that GATA4 protects these malignancies from TRAIL-induced apoptosis. GATA4 presumably exerts this effect by regulating the expression of anti-apoptotic Bcl-2. This is of particular interest as high expression of GATA4 is known to correlate to aggressive GCT behavior. Thus, GATA4 seems to protect GCTs from endogenous TRAIL by upregulating anti-apoptotic factors such as Bcl-2.

Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells

In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor PI, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.

Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries. © 2010 Elsevier Inc.

Agonista versus antagonista do GnRH em ciclos de reprodução assistida: DNA fragmentação e apoptose das células da granulosa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Incomplete ovariosalpingectomy and subsequent malignant granulosa cell tumor in a female green iguana (Iguana iguana)

Case Description-A 9-year-old spayed female green iguana (Iguana iguana) was evaluated because of a distended coelom and weight loss. History included a single episode of egg binding and subsequent bilateral ovariosalpingectomy.;Clinical Findings-Physical examination revealed a mass within the coelomic cavity. Ultrasonography revealed a large, irregular mass with hypoechoic regions and coelomic effusion. Clinicopathologic derangements included heterophilia, monocytosis, lymphopenia, basophilia, hypocholesterolemia, hypoproteinemia, and hypercalcemia. Results of cytologic evaluation of the mass were suggestive of malignant epithelial neoplasia, but neoplastic cells were not found in the effusion. An ovarian tumor was suspected on the basis of clinical signs, clinicopathologic findings, and results of cytologic evaluation of the mass.;Treatment and Outcome-Surgical exploration revealed a large left ovary, a normal-appearing contralateral ovary, and a mass in the fat body, all of which were removed and submitted for histologic examination. The histologic diagnosis was granulosa cell tumor with metastasis to the fat body. The patient died 11 months after evaluation, and disseminated granulosa cell tumor was confirmed at necropsy; histologic examination at that time also identified systemic mastocytosis.;Clinical Relevance-Granulosa cell tumors are uncommon in reptiles, and this was the first granulosa cell tumor described antemortem cytologically, histologically, and ultrastructurally in an iguana. Findings in this iguana underscored concerns associated with incomplete oophorectomy of iguanas; cytologic and histopathologic findings were similar to those observed in other domestic animals. Oophorectomy should be considered as an alternative to standard ovariosalpingectomy to avoid potential complications in pet reptiles, and use of microsurgical instruments and vascular clips is advised. (J Am Vet Med Assoc 2011239:237-242)

Expression profiling of purified mouse gonadal somatic cells during the critical time window of sex determination reveals novel candidate genes for human sexual dysgenesis syndromes

Despite the identification of SRY as the testis-determining gene in mammals, the genetic interactions controlling the earliest steps of male sex determination remain poorly understood. In particular, the molecular lesions underlying a high proportion of human XY gonadal dysgenesis, XX maleness and XX true hermaphroditism remain undiscovered. A number of screens have identified candidate genes whose expression is modulated during testis or ovary differentiation in mice, but these screens have used whole gonads, consisting of multiple cell types, or stages of gonadal development well beyond the time of sex determination. We describe here a novel reporter mouse line that expresses enhanced green fluorescent protein under the control of an Sf1 promoter fragment, marking Sertoli and granulosa cell precursors during the critical period of sex determination. These cells were purified from gonads of male and female transgenic embryos at 10.5 dpc (shortly after Sry transcription is activated) and 11.5 dpc (when Sox9 transcription begins), and their transcriptomes analysed using Affymetrix genome arrays. We identified 266 genes, including Dhh, Fgf9 and Ptgds, that were upregulated and 50 genes that were downregulated in 11.5 dpc male somatic gonad cells only, and 242 genes, including Fst, that were upregulated in 11.5 dpc female somatic gonad cells only. The majority of these genes are novel genes that lack identifiable homology, and several human orthologues were found to map to chromosomal loci implicated in disorders of sexual development. These genes represent an important resource with which to piece together the earliest steps of sex determination and gonad development, and provide new candidates for mutation searching in human sexual dysgenesis syndromes.