Solange Magre

Contribution of Germ Cells to the Differentiation and Maturation of the Ovary: Insights from Models of Germ Cell Depletion

Abstract In mammals, the role played by germ cells in ovarian differentiation and folliculogenesis has been the focus of an increasing number of studies over the last decades. From these studies, it has emerged that bidirectional communication between germ cells and surrounding companion cells is required as soon as the initial assembly of follicles. Models of germ cell depletion that arise from both spontaneous and experimentally induced mutations as well as irradiation or chemical treatments have been helpful in deciphering the role played by germ cells from the onset of ovarian differentiation onward. This review reports current knowledge and proposes novel hypotheses that can be formulated from these models about the contribution of germ cells to ovarian differentiation and folliculogenesis. In particular, it promotes the idea that the influence of germ cells on companion somatic cells varies within both ovarian differentiation and folliculogenesis.

EVIDENCE THAT GONADOTROPIN-RELEASING HORMONE STIMULATES GENE EXPRESSION AND LEVELS OF ACTIVE NITRIC OXIDE SYNTHASE TYPE I IN PITUITARY GONADOTROPHS, A PROCESS ALTERED BY DESENSITIZATION AND, INDIRECTLY, BY GONADAL STEROIDS

To determine the site and mechanism of action of gonadal steroids on pituitary nitric oxide synthase type I (NOS I), present in both gonadotrophs and folliculo-stellate cells, the effects of castration and steroids were examined in male rats, in the presence of a GnRH antagonist (Antarelix). Western analysis showed a rapid and substantial increase with time, after orchidectomy, of NOS I protein, the concentration doubling in 24 h and reaching a maximal 4- to 5-fold increase after 3–7 days, followed by a progressive decline after 2 weeks. Testosterone or estradiol replacement, or administration of GnRH antagonist, totally abolished the effects of castration, demonstrating a mediation of the steroid effects via GnRH. In noncastrated rats, steroids and the GnRH antagonist also caused a reduction in the levels of NOS I (by 50–60%), consistent with inhibition of endogenous GnRH stimulation. In marked contrast, administration of a potent GnRH agonist (Triptorelin) to intact rats increased the levels of NOS I. A...

GnRH Receptor Gene Expression in the Developing Rat Hippocampus: Transcriptional Regulation and Potential Roles in Neuronal Plasticity

In the pituitary of mammals, the GnRH receptor (GnRHR) plays a primary role in the control of reproductive function. It is further expressed in the hippocampus, where its function, however, is not well defined. By quantitative RT-PCR analyses, we demonstrate herein that the onset of GnRHR gene (Gnrhr) expression in the rat hippocampus was unexpectedly delayed as compared to the pituitary and only occurred after birth. Using a previously described transgenic mouse model bearing the human placental alkaline phosphatase reporter gene under the control of the rat Gnrhr promoter, we established a positive correlation between the temporal pattern of Gnrhr mRNA levels and promoter activity in the hippocampal formation. The gradual appearance of human placental alkaline phosphatase transgene expression occurred simultaneously in the hippocampus and interconnected structures such as the lateral septum and the amygdala, coinciding with the establishment of hippocampo-septal projections. Analysis of transcription factors together with transient transfection assays in hippocampal neurons indicated that the combinatorial code governing the hippocampus-specific expression of the Gnrhr is distinct from the pituitary, likely involving transactivating factors such as NUR77, cyclic AMP response element binding protein, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene homolog. A silencing transcription factor acting via the -3255/-1135 promoter region of the Gnrhr may be responsible for the transcriptional repression observed around birth. Finally, GnRH directly stimulated via activation of its receptor the expression of several marker genes of neuronal plasticity such as Egr1, synaptophysin, and spinophilin in hippocampal primary cultures, suggesting a role for GnRHR in neuronal plasticity. Further characterization of these mechanisms may help unravel important functions of GnRH/GnRHR signaling in the brain.

What is the role of PACAP in gonadotrope function

Strong evidence in favor of a direct action of hypothalamic PACAP at the pituitary to modulate gonadotrope function has been acquired mainly by in vitro studies using cultured pituitary cells or gonadotrope cell lines. In particular, PACAP has been shown to cooperate with GnRH, the primary regulator of gonadotropes, to regulate/modulate gonadotropin subunit gene expression, gonadotropin release as well as gonadotrope responsiveness. These effects of PACAP appear to be due essentially to its high potent stimulatory action on the cAMP/protein kinase pathway. Ensuing mechanisms include signaling cross-talk and/or enhanced gene expression within gonadotropes. PACAP may also indirectly operate on these cells through paracrine mechanisms. While PACAP has long been viewed as a hypophysiotropic factor, a locally produced PACAP has also been described. Interestingly, both appear similarly up-regulated at proestrus of the reproductive cycle in female rats. Further in vivo investigation is now necessary to ascertain the physiological relevance of the observed pituitary PACAP effects and especially to evaluate the respective contribution of hypothalamic and pituitary PACAP in the dynamic control of gonadotrope function.

Oocyte-specific inactivation of Omcg1 leads to DNA damage and c-Abl/TAp63-dependent oocyte death associated with dramatic remodeling of ovarian somatic cells

Aberrant loss of oocytes following cancer treatments or genetic mutations leads to premature ovarian insufficiency (POI) associated with endocrine-related disorders in 1% of women. Therefore, understanding the mechanisms governing oocyte death is crucial for the preservation of female fertility. Here, we report the striking reproductive features of a novel mouse model of POI obtained through oocyte-specific inactivation (ocKO) of Omcg1/Zfp830 encoding a nuclear zinc finger protein involved in pre-mRNA processing. Genetic ablation of OMCG1 in early growing oocytes leads to reduced transcription, accumulation of DNA double-strand breaks and subsequent c-Abl/TAp63-dependent oocyte death, thus uncovering the key role of OMCG1 for oocyte genomic integrity. All adult Omcg1ocKO females displayed complete elimination of early growing oocytes and sterility. Unexpectedly, mutant females exhibited a normal onset of puberty and sexual receptivity. Detailed studies of Omcg1ocKO ovaries revealed that the ovarian somatic compartment underwent a dramatic structural and functional remodeling. This allowed the cooperation between oocyte-depleted follicles and interstitial tissue to produce estradiol. Moreover, despite early folliculogenesis arrest, mutant mice exhibited sexual cyclicity as shown by cyclical changes in estrogen secretion, vaginal epithelium cytology and genital tract weight. Collectively, our findings demonstrate the key role of Omcg1 for oocyte survival and highlight the contribution of p63 pathway in damaged oocyte elimination in adulthood. Moreover, our findings challenge the prevailing view that sexual cyclicity is tightly dependent upon the pace of folliculogenesis and luteal differentiation.

Gender Differences in Transcriptional Signature of Developing Rat Testes and Ovaries following Embryonic Exposure to 2,3,7,8-TCDD

Dioxins are persistent organic pollutants interfering with endocrine systems and causing reproductive and developmental disorders. The objective of our project was to determine the impact of an in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on reproductive function of male and female offspring in the rat with a special emphasis on the immature period. We used a low dose of TCDD (unique exposure by oral gavage of 200 ng/kg at 15.5 days of gestation) in order to mirror a response to an environmental dose of TCDD not altering fertility of the progeny. We choose a global gene expression approach using Affymetrix microarray analysis, and testes of 5 days and ovaries of 14 days of age. Less than 1% of the expressed genes in gonads were altered following embryonic TCDD exposure; specifically, 113 genes in ovaries and 56 in testes with 7 genes common to both sex gonads. It included the repressor of the aryl hydrocarbon receptor (Ahrr), the chemokines Ccl5 and Cxcl4 previously shown to be regulated by dioxin in testis, Pgds2/Hpgds and 3 others uncharacterized. To validate and extend the microarray data we realized real-time PCR on gonads at various developmental periods of interest (from 3 to 25 days for ovaries, from 5 to the adult age for testes). Overall, our results evidenced that both sex gonads responded differently to TCDD exposure. For example, we observed induction of the canonic battery of TCDD-induced genes coding enzymes of the detoxifying machinery in ovaries aged of 3–14 days of age (except Cyp1a1 induced at 3–10 days) but not in testes of 5 days (except Ahrr). We also illustrated that inflammatory pathway is one pathway activated by TCDD in gonads. Finally, we identified several new genes targeted by TCDD including Fgf13 in testis and one gene, Ptgds2/Hpgds regulated in the two sex gonads.

OVEX1, a novel chicken endogenous retrovirus with sex-specific and left-right asymmetrical expression in gonads

BackgroundIn chickens, as in most birds, female gonad morphogenesis is asymmetrical. Gonads appear first rather similarly, but only the left one undergoes full differentiation and gives rise to a functional ovary. The right gonad, in which the cortex does not develop, remains restricted to the medulla and finally regresses. Opportunity was taken of this left-right asymmetry to perform a suppression subtractive hybridization screening to select for transcripts preferentially expressed in the developing left ovary as compared to the right one, and thus identify genes that are potentially involved in the process of ovarian differentiation.ResultsOne of these transcripts, named Ovex1 according to its expression profile, corresponds to an endogenous retrovirus that has not been previously characterized. It is transcribed as full-length and singly spliced mRNAs and contains three uninterrupted open reading frames coding potentially for proteins with homology to Gag and Pro-Pol retroviral polyproteins and a third protein showing only a weak similarity with Env glycoproteins. Ovex1 is severely degenerated; it is devoid of typical long terminal repeats and displays some evidence of recombination. An orthologous Ovex1 locus was identified in the genome of zebra finch, a member of a different bird order, and similar sequences were detected in turkey, guinea fowl, and duck DNA. The relationship between these sequences follows the bird phylogeny, suggesting vertical transmission of the endogenous retrovirus for more than 100 million years.Ovex1 is transcribed in chicken gonads with a sex-dependent and left-right asymmetrical pattern. It is first expressed in the cortex of the left indifferent gonads of both sexes. Expression is transient in the left testis and absent in the right one. In developing ovaries, Ovex1 transcription increases sharply in the left cortex and is weakly detected in the medulla. After folliculogenesis, Ovex1-expressing cells constitute the follicular granulosa cell layer. Ovex1 expression highlights a striking desquamation process that leads to profound cortical remodeling associated with follicle morphogenesis.ConclusionEvidence for a selection pressure at the protein level suggests that this endogenous retrovirus, expressed in the ovarian supporting cell lineage, might play an active role in bird ovarian physiology.

A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty

In cyclic females, FSH stimulates ovarian estradiol (E2) production and follicular growth up to the terminal stage. A transient elevation in circulating FSH and E2 levels occurs shortly after birth. But what could be the action of FSH on the ovary during this period, and in particular how it stimulates ovarian steroidogenesis without supporting terminal follicular maturation is intriguing. By experimentally manipulating FSH levels, we demonstrate in mice that the mid-infantile elevation in FSH is mandatory for E2 production by the immature ovary, but that it does not stimulate follicle growth. Importantly, FSH increases aromatase expression to stimulate E2 synthesis, however it becomes unable to induce cyclin D2, a major driver of granulosa cell proliferation. Besides, although FSH prematurely induces luteinizing hormone (LH) receptor expression in granulosa cells, LH pathway is not functional in these cells to induce their terminal differentiation. In line with these results, supplying infantile mice with a superovulation regimen exacerbates E2 production, but it does not stimulate the growth of follicles and it does not induce ovulation. Overall, our findings unveil a regulation whereby high postnatal FSH concentrations ensure the supply of E2 required for programming adult reproductive function without inducing follicular maturation before puberty.

Reporter transgenic mouse models highlight the dual endocrine and neural facet of GnRH receptor function

In the pituitary of mammals, the GnRH receptor (GnRHR) plays crucial roles in the neuroendocrine control of reproductive function. This receptor is specifically expressed by the gonadotrope cells scattered among the five other endocrine cell types constituting the anterior pituitary; it is also expressed in other organs, such as the gonads and brain where its function is not well defined. To gain insight into GnRHR function, distribution, and regulation, several transgenic approaches have been developed using a range of reporter genes under the control of the mouse, rat, or ovine GnRHR gene (Gnrhr) promoters. Comprehensive reviews of the literature, together with recent results obtained in our laboratory, illustrate how these transgenic models highlight the endocrine as well as the neural facet of GnRHR function. In this review, the endocrine aspect will be discussed with regard to the pituitary and gonad function, whereas the neural aspect will be discussed with regard to hippocampal formation and the oculomotor pathway, the latter constituting an unpreviously described site of Gnrhr promoter activity. These approaches should help elucidate the properties of the mammalian GnRH system.

The GnRH receptor and the response of gonadotrope cells to GnRH pulse frequency code. A story of an atypical adaptation of cell function relying on a lack of receptor homologous desensitization.

Brain control of the reproductive system is mediated through hypothalamic gonadotropin-releasing hormone (GnRH) which activates specific receptors (GnRHR) present at the surface of the pituitary gonadotropes to trigger secretion of the two gonadotropins LH and FSH. A unique feature of this system is the high dependence on the secretion mode of GnRH, which is basically pulsatile but undergoes considerable fluctuations in pulse frequency pattern in response to endogenous or external factors. How the physiological fluctuations of GnRH secretion that orchestrate normal reproduction are decoded by the gonadotrope cell machinery to ultimately control gonadotropin release and/or subunit gene transcription has been the subject of intensive studies during the past decades. Surprisingly, the mammalian GnRHR is unique among G protein-coupled receptor family as it lacks the carboxy-terminal tail usually involved in classical endocytotic process. Accordingly, it does not desensitize properly and internalizes very poorly. Both this atypical intrinsic property and post-receptor events may thus contribute to decode the GnRH signal. This includes the participation of a network of signaling pathways that differently respond to GnRH together with a growing amount of genes differentially sensitive to pulse frequency. Among these are two pairs of genes, the transcription factors EGR-1 and NAB, and the regulatory factors activin and follistatin, that function as intracellular autoregulatory feedback loops controlling respectively LHbeta and FSHbeta gene expression and hence, LH and FSH synthesis. Pituitary gonadotropes thus represent a unique model of cells functionally adapted to respond to a considerably fluctuating neuroendocrine stimulation, from short individual pulses to sustained GnRH as observed at the proestrus of ovarian cycle. Altogether, the data emphasize the adaptative reciprocal complementarity of hypothalamic GnRH neurones and pituitary gonadotropes to function as an original unit.