Justine Bouilly

Impact of prolactin receptor isoforms on reproduction

Prolactin is a hormone involved in growth, development, reproduction, metabolism, water and electrolyte balance, brain and behavior, and immunoregulation. Its actions on reproductive processes represent the largest group of functions identified for this hormone. Besides the classic long form of the prolactin receptor, many short form receptors have been identified in rodents and human tissues. Mouse mutagenesis studies have offered insight into the biology of the prolactin family, providing compelling evidence that different isoforms have independent biological activity. The possibility that short forms mediate cell proliferation is important for a variety of tissues including mammary glands and ovarian follicles. This review summarizes the current knowledge about prolactin signaling and its role in reproduction through either long or short isoform receptors.

Hyperprolactinemia-induced ovarian acyclicity is reversed by kisspeptin administration

Hyperprolactinemia is the most common cause of hypogonadotropic anovulation and is one of the leading causes of infertility in women aged 25-34. Hyperprolactinemia has been proposed to block ovulation through inhibition of GnRH release. Kisspeptin neurons, which express prolactin receptors, were recently identified as major regulators of GnRH neurons. To mimic the human pathology of anovulation, we continuously infused female mice with prolactin. Our studies demonstrated that hyperprolactinemia in mice induced anovulation, reduced GnRH and gonadotropin secretion, and diminished kisspeptin expression. Kisspeptin administration restored gonadotropin secretion and ovarian cyclicity, suggesting that kisspeptin neurons play a major role in hyperprolactinemic anovulation. Our studies indicate that administration of kisspeptin may serve as an alternative therapeutic approach to restore the fertility of hyperprolactinemic women who are resistant or intolerant to dopamine agonists.

Novel NOBOX loss‐of‐function mutations account for 6.2% of cases in a large primary ovarian insufficiency cohort

Primary ovarian insufficiency (POI) is a disorder associated with female infertility, which affects approximately 1% of women under 40 years of age. A genetic component has been suggested as one possible cause of the majority of cases of nonsyndromic forms. Newborn Ovary Homeobox (NOBOX) is an ovary‐specific gene, playing a critical role in ovary in mice, as its absence leads to sterility mimicking a POI. In this study, we sequenced NOBOX in a cohort of 178 women with idiopathic POI. Among 19 identified variations, we described one nonsense (c.907C>T/p.R303X) and four missense (c.271G>T/p.G91W, c.349C>T/p.R117W, c.1025G>C/p.S342T, and c.1048G>T/p.V350L) NOBOX heterozygous mutations in 12 patients. We reproduced each of the five mutations and tested their effects on the signaling activity in transfected cells. We demonstrated that these mutations compromised the ability of the proteins to bind to and transactivate the well‐known growth differentiation factor 9 (GDF9) promoter. The pattern of our findings suggests that the genetic mechanism in humans responsible for POI in women involves haploinsufficiency rather than dominant negative gene action. The identification, characterization, and the very high 6.2% prevalence of these new mutations in POI patients suggest considering NOBOX as the first autosomal candidate gene involved in this syndrome. Hum Mutat 32:1108–1113, 2011. ©2011 Wiley‐Liss, Inc.

Prolactin signaling mechanisms in ovary.

Prolactin is a hormone that is essential for normal reproduction and signals through two types of receptors. Not only is the classical long form of the prolactin receptor identified, but so are many short form receptors in rodents and human tissues. Mouse mutagenesis studies have offered insight into the biology of prolactin family, providing compelling evidence that the different isoforms have independent biological activity. The possibility that short forms mediate cell proliferation is important for a variety of tissues including mammary gland and ovarian follicles. This review summarizes our current knowledge about prolactin signaling and its role in reproduction through either long or short isoform receptors.

New NOBOX Mutations Identified in a Large Cohort of Women With Primary Ovarian Insufficiency Decrease KIT-L Expression

CONTEXT Primary ovarian insufficiency (POI) is a major cause of anovulation and infertility in women. This disease affects 1% of women before 40 years, and several genetic causes have been reported. OBJECTIVE The aim of the study was to evaluate the prevalence of NOBOX mutations in a new large cohort of women with POI and to characterize these variants and identify a NOBOX novel target gene. PATIENTS AND METHODS A total of 213 unrelated patients with POI were screened for NOBOX mutations, and luciferase reporter assays were performed for the mutations identified. RESULTS We reported 3 novel and 2 recurrent heterozygous missense NOBOX rare variants found in 12 patients but not in 724 alleles from ethnic-matched individual women with occurrence of menopause at a normal age. Their functional impact had been tested on the classic growth differentiation factor-9 (GDF9) promoter and on KIT-L, a new NOBOX target gene. The p.Gly91Thr, p.Gly111Arg, p.Arg117Trp, p.Lys371Thr, and p.Pro619Leu mutations were deleterious for protein function. CONCLUSIONS In our series, 5.6% of the patients with POI displayed heterozygous NOBOX mutations. We demonstrate that KIT-L could be now a direct NOBOX target. These findings replicate the high prevalence of the association between the NOBOX rare variants and POI.

NOBOX is a key FOXL2 partner involved in ovarian folliculogenesis

Dear Editor, Early ovarian expression of Newborn oogenesis homeobox (NOBOX), a transcription factor belonging to the homeoprotein class, is decisive for the progression of primordial follicles to the primary follicular stage as demonstrated by the sterility of Nobox 2 female mice (Rajkovic et al., 2004). Consequently, the loss of the Nobox gene causes primary ovarian insufficiency (POI) (Rajkovic et al., 2004) and we have shown that 6.2% of POI cases harbor mutations in the NOBOX gene (Qin et al., 2007; Bouilly et al., 2011). Such a high prevalence of mutations demonstrates that NOBOX is a major autosomal gene involved in POI (Bouilly et al., 2011). However, the exact role of this transcription factor during folliculogenesis is unclear. The forkhead transcription factor 2 (Foxl2) is a central element in ovarian development and follicular growth and maturation. FOXL2 mutations are responsible for blepharophimosis-ptosis-epicantus inversus syndrome (BPES), often associated with POI (Moumne et al., 2008; Beysen et al., 2009). Lack of Foxl2 during development leads to the absence of the secondary follicles and no differentiation of squamous granulosa cells (FGCs) into cuboidal cell (Schmidt et al., 2004; Uda et al., 2004). However, little is known about the mechanism by which Foxl2 regulates different target gene subsets depending on developmental stages and/or other signals. Such target specification may be achieved through interactions with protein partners modulating promoter recognition. Homeoproteins and Forkhead transcription factors are protein families characterized by conserved DNA-binding domains. Interestingly, it has been documented that homeoproteins can interact with Forkhead box transcription factors giving rise to generic ‘inter-family’ interactions (Foucher et al., 2003). In this study, we show that NOBOX, initially described as expressed in the oocyte (Rajkovic et al., 2004), is also expressed in FGCs. Besides, we identify NOBOX as a key FOXL2 partner and report the existence of the first ovarian homeoprotein/forkhead box complex to regulate pivotal ovarian genes involved in folliculogenesis. Although NOBOX is undoubtedly involved in the etiology of POI, only limited information regarding its regulators is available. To further explore the localization of the NOBOX protein, we have performed an immunohistological examination of ovarian sections from human fetuses revealing the presence of a strong staining of oogonia and also future follicular cells (Figure 1A and B). A similar analysis performed on ovarian sections of a 14-year girl showed that NOBOX was present in oocytes of primordial and primary follicles grouped in nests in the ovarian cortex (Figure 1C). Unexpectedly, primordial, primary, and secondaryfollicles also showed NOBOX labeling in most FGCs (Figure 1C and D), which was confirmed by RT–PCR in human FGCs (Figure 1E), suggesting a new ovarian role for this protein. Taking into account the fact that both NOBOX and FOXL2 are co-expressed, and that homeodomain/forkhead generic interactions can take place (Foucher et al., 2003), we hypothesized that they could be partners in the regulation of transcriptional targets. To validate this hypothesis, we showed that in transfected cells, NOBOX binds to FOXL2 to form a stable complex resisting co-immunoprecipitation (Figure 1F). To confirm the NOBOX–FOXL2 interaction, we used a mammalian two-hybrid system where the readout is a luciferase activity. In agreement with our co-immunoprecipitation data, we detected a significant increase of luciferase activity after co-overexpression of both proteins, confirming the interaction between NOBOX and FOXL2 (Supplementary Figure S1A). Based on the suggestion that forkhead domains and homeoboxes can foster generic interactions, we performed a two-hybrid assay using the relevant domains. A significantly increased luciferase activity demonstrated that NOBOX and FOXL2 interact via their DNA-binding domains (Supplementary Figure S1B). FOXL2 has been shown to activate its own promoter (hereafter named DK3 promoter) (Benayoun et al., 2008), containing three FOXL2 response elements (FLRE). This promoter also contains two NOBOX binding elements (NBE) as determined in silico. To assess the impact of the interaction of NOBOX and FOXL2 on their function, we tested the activity of NOBOX and FOXL2 on the DK3 promoter. Overexpression of either NOBOX or FOXL2 alone led to a significant transactivation of the promoter (P , 0.01). However, the co-overexpression of both transcription factors led to a significantly decreased activity with respect to the levels observed when each factor was overexpressed alone (Figure 1G). We confirmed these results with another reporter. Indeed, the OCT4 promoter, although ‘artificial’ because OCT4 is not expressed in FGCs, proved to be informative in molecular terms (Choi and Rajkovic, 2006) (Supplementary Figure S2A). Thus, we also examined whether the presence of the NOBOX and FOXL2 binding sites in the OCT4 promoter were involved in mediating their effects on transcription. NOBOX was, as expected, unable to activate the OCT4 promoter bearing a mutated NBE (Supplementary Figure S2B). Similarly, FOXL2 was unable to activate the promoter mutated for the FLRE (Supplementary Figure S2C). Surprisingly, the co-transfection of both factors represses the transcriptional activity of the mutated versions of OCT4 (Supplementary Figure S2B doi:10.1093/jmcb/mju006 Journal of Molecular Cell Biology (2014), 6, 175–177 | 175 Published online March 11, 2014

Ovarian function is restored after grafting of cryopreserved immature ovary in ewes

As a result of advances in medical treatment, almost 80% of children who are diagnosed with cancer survive long‐term. The adverse consequences of cancer treatments include impaired puberty and fertility. In prepubertal girls, the only therapeutic option is the cryopreservation of an ovary. To date, a dozen births have been reported after reimplantation of cryopreserved mature ovaries. To analyze ovarian function after immature grafts, we performed ovarian grafting in a ewe model. Fresh or cryopreserved ovaries from immature ewes were autografted in prepubertal or adult ewes. Cyclic hormonal activity was recovered 3 mo after grafting. Histological analysis demonstrated the presence of all follicle populations and corpora lutea not affected by cryopreservation. After 3 reproductive seasons, births had been observed in all groups, and the follicle‐stimulating hormone status was under the limit, which indicated an exhausted ovary. As an indicator of potential imprinting default, the methylation status of the Igf2r gene was analyzed and did not show significant alteration compared with that of nonmanipulated animals. Taken together, these results demonstrate that immature ovarian grafting is able to restore spontaneous puberty and fertility and could guide the reimplantation of immature cortex in women.—Sauvat, F., Bouilly, J., Capito, C., Lefèvre, A., Blachère, T., Borenstein, N., Sarnacki, S., Dandolo, L., Binart, N. Ovarian function is restored after grafting of cryopreserved immature ovary in ewes. FASEB J. 27, 1511–1518 (2013). www.fasebj.org

Impaired protein stability and nuclear localization of NOBOX variants associated with premature ovarian insufficiency

Premature ovarian insufficiency (POI) is a clinical syndrome defined by a loss of ovarian activity before the age of 40. Its pathogenesis is still largely unknown, but increasing evidences support a genetic basis in most cases. Among these, heterozygous mutations in NOBOX, a homeobox gene encoding a transcription factor expressed specifically by oocyte and granulosa cells within the ovary, have been reported in ∼6% of women with sporadic POI. The pivotal role of NOBOX in early folliculogenesis is supported by findings in knock-out mice. Here, we report the genetic screening of 107 European women with idiopathic POI, recruited in various settings, and the molecular and functional characterization of the identified variants to evaluate their involvement in POI onset. Specifically, we report the identification of two novel and two recurrent heterozygous NOBOX variants in 7 out of 107 patients, with a prevalence of 6.5% (upper 95% confidence limit of 11.17%). Furthermore, immunolocalization, Western Blot and transcriptional assays conducted in either HEK293T or CHO cells revealed that all the studied variants (p.R44L, p.G91W, p.G111R, p.G152R, p.K273*, p.R449* and p.D452N) display variable degrees of functional impairment, including defects in transcriptional activity, autophagosomal degradation, nuclear localization or protein instability. Several variants conserve the ability to interact with FOXL2 in intracellular aggregates. Their inability to sustain gene expression, together with their likely aberrant effects on protein stability and degradation, make the identified NOBOX mutations a plausible cause of POI onset.

Germline Prolactin Receptor Mutation Is Not a Major Cause of Sporadic Prolactinoma in Humans

Background/Aims: No genetic anomalies specifically predisposing humans to prolactinomas have so far been identified. The prolactin receptor (PRLR) is a good candidate, however, as Prlr knockout mice develop prolactinomas, and a case of familial hyperprolactinemia has been linked to PRLR mutation. The main objective of this study was to detect germline PRLR mutations in patients with sporadic prolactinomas unrelated to AIP or MEN1 mutation. Methods: We sequenced all PRLR exons and intron-exon junctions on genomic DNA from 88 patients with a median age of 24 years. Results: We identified 4 PRLR variations (p.Ile76Val, p.Ile146Leu, p.Glu108Lys and p.Glu554Gln) in 16 patients. One patient had the rare variant p.Glu554Gln in the heterozygous state. Another patient had the extremely rare p.Glu108Lys variant described here for the first time. The other 2 variants (p.Ile76Val and p.Ile146Leu) are relatively common in the general population. All these 4 variants have been functionally tested in vitro and have no effect on PRLR expression, localization and signaling after prolactin stimulation. Conclusion: Inactivating germline variations of PRLR are not associated with sporadic prolactinoma in this series. Nevertheless, somatic disruption of PRLR has not been excluded in this subset of pituitary tumors.

Sequence variation analysis of the prolactin receptor C-terminal region in women with premature ovarian failure

Using a mouse model expressing only the PRL receptor short isoform mimicking premature ovarian failure, signaling pathways induced by PRL were analyzed in mouse ovaries. Sequencing of the coding portion of exons 10 and 11, specific to the long and short receptor isoform, respectively, did not revealed any mutation in 101 women with premature ovarian failure.