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1.
Cell Death Differ ; 22(1): 108-17, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25168238

ABSTRACT

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 Omcg1(ocKO) 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 Omcg1(ocKO) 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.


Subject(s)
Cell Cycle Proteins/metabolism , Genomic Instability/physiology , Nuclear Proteins/metabolism , Oocytes/metabolism , Ovary/metabolism , Phosphoproteins/metabolism , Trans-Activators/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Death , Cell Survival , Estrogens/metabolism , Female , Male , Mice , Mice, Knockout , Nuclear Proteins/genetics , Oocytes/cytology , Ovary/cytology , Phosphoproteins/genetics , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Trans-Activators/genetics
2.
Int J Androl ; 33(2): 413-24, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20059583

ABSTRACT

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like compounds are widely encountered toxic substances suspected of interfering with the endocrine systems of humans and wildlife, and of contributing to the loss of fertility. In this study, we determined the changes in testicular gene expression caused by in utero exposure to TCDD along with the intra-testicular testosterone levels, epididymal sperm reserves, daily sperm production (DSP) and testis histology. To this purpose, female pregnant Sprague-Dawley rats orally received TCDD (10, 100 or 200 ng/kg body weight) or vehicle at embryonic day 15, and the offspring was killed throughout development. Hepatic Cyp1a1 gene expression was measured in the offspring to confirm the exposure to TCDD. The gross histology of the testes and intra-testicular testosterone levels were normal among the studied groups. Sperm reserves were altered in 67-day-old rats of the TCDD-200 group, but not in 145-day-old animals or in the other TCDD-exposed groups. Nonetheless, fertility was not altered in males of the TCDD-200 group, and the F2 males generated had normal sperm reserves and DSP. Microarray analysis permitted the identification of eight differentially expressed genes in the 4-week-old testes of the TCDD-200 compared with that of the control group (cut-off value +/- 1.40), including the down-regulated chemokine Ccl5/Rantes. Inhibition of Ccl5/Rantes gene expression was observed throughout development in the TCDD-200 group, and at 67 and 145 days in the TCDD-100 group (animals of younger ages were not examined). Ccl5/Rantes gene expression was mostly confined in Leydig cells. F2 males generated from males of the TCDD-200 group had normal levels of Ccl5/Rantes in testis and Cyp1a1 in liver, which might indicate that Ccl5/Rantes is a marker of TCDD exposure in testis such as Cyp1a1 in liver. In conclusion, we demonstrated a decrease in Ccl5/Rantes RNA levels and a transitory decline in sperm reserves in the testes of rats of TCDD-dosed dams.


Subject(s)
Chemokine CCL5/genetics , Polychlorinated Dibenzodioxins/toxicity , Prenatal Exposure Delayed Effects/pathology , Reproduction/drug effects , Testis/drug effects , Animals , Cytochrome P-450 CYP1A1/metabolism , Down-Regulation , Female , Liver/enzymology , Male , Maternal Exposure , Pregnancy , Rats , Rats, Sprague-Dawley , Sperm Count , Testis/metabolism
3.
J Endocrinol ; 187(1): 135-47, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16214949

ABSTRACT

The role of fibroblast growth factor (FGF) 2 and FGF9 as mediators of cell-cell interactions between Sertoli cells (SCs) and peritubular cells (PCs) was investigated. Using RT-PCR, we demonstrated that SCs and PCs recovered from 20-day-old rats expressed several of the seven FGF receptors (FGFRs), and more specifically the FGFR1 IIIc. FGF2 and FGF9 did not elicit any morphological changes in primary cultures of SCs, nor did they alter the number of SCs in culture. By contrast, changes in shape were observed in FGF2- and FGF9-treated PCs. In addition, FGF2 but not FGF9 enhanced significantly and dose-dependently the number of PCs in culture, indicating that FGF2 was a survival factor for these cells. It was also mitogenic because it enhanced the [3H]thymidine labeling index in PCs. We next examined the effects of FGF2 and FGF9 in a coculture system using 20-day-old rat SCs and PCs, and in an organotypic culture system using XY rat embryonic gonads. In both models, FGF2 and FGF9 were found to promote cellular interactions as evidenced by the extent of cellular reorganization in the coculture system, and cord morphogenesis and growth in the organotypic culture system. A key feature in SC-PC interactions is the synthesis and remodeling of the basement membrane which is co-elaborated by the two cell types. Since basement membrane homeostasis depends upon the coordinated activity of proteinases and inhibitors, the proteinases and inhibitors produced by PCs and SCs degrading or opposing degradation of the major components of the basement membrane were further studied. Specifically, we monitored the metalloproteinases (MMP)-2 and -9 and the tissue inhibitors -1, -2 and -3, the plasminogen activators (PAs) and the PA inhibitor-1, using zymography for the proteinases and Western blots for the cognate inhibitors. Cocultures received FGF or an analog of cAMP in order to prevent cellular reorganization. We found that FGF2 was unique in inducing MMP-9 in coculture. Also, the enhanced levels of the PA inhibitor-1 and the 30 kDa band glycosylated form of tissue inhibitor-3 correlated with the enhanced SC-PC reorganization. It was concluded that FGF2 and FGF9 are morphogens for the formation of testicular cords.


Subject(s)
Epithelial Cells/cytology , Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factor 9/pharmacology , Mesoderm/cytology , Testis/embryology , Animals , Cell Division/drug effects , Cell Shape/drug effects , Cells, Cultured , Coculture Techniques , DNA/biosynthesis , Epithelial Cells/drug effects , Male , Mesoderm/drug effects , Organ Culture Techniques , Rats , Reverse Transcriptase Polymerase Chain Reaction , Seminiferous Tubules/cytology , Seminiferous Tubules/drug effects , Sertoli Cells/cytology , Sertoli Cells/drug effects , Testis/drug effects
4.
Rev Sci Tech ; 24(1): 323-34, 2005 Apr.
Article in English | MEDLINE | ID: mdl-16110899

ABSTRACT

Xenotransplantation, in particular the transplantation of pig cells, tissues and organs into human recipients, may alleviate the current shortage of suitable allografts available for human transplantation. This overview addresses the physiological, immunological and microbial factors involved in xenotransplantation. The issues reviewed include the merits of using pigs as xenograft source species, the compatibility of pig and human organ physiology, and the rejection mechanism and attempts to overcome this immunological challenge. The authors discuss advances in the prevention of pig organ rejection through the creation of genetically modified pigs, more suited to the human micro-environment. Finally, in regard to microbial hazards, the authors review possible viral infections originating from pigs.


Subject(s)
Swine Diseases/transmission , Swine/anatomy & histology , Swine/physiology , Transplantation, Heterologous/adverse effects , Virus Diseases/veterinary , Animals , Graft Rejection , Humans , Swine/virology , Virus Diseases/transmission , Zoonoses
6.
Gene Expr Patterns ; 2(3-4): 373-7, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12617828

ABSTRACT

Lhx9 (LIM/Homeobox gene 9) encodes a transcription factor implicated in various developmental processes, including gonadogenesis. Our observations in the rat show that Lhx9 expression present in undifferentiated gonads disappears as epithelial cells differentiate into Sertoli cells and begin to express AMH. In rat and in chick testes, Lhx9 expression present in interstitial cells decreases progressively to become undetectable after birth. In the female rat, Lhx9 is highly expressed in epithelial ovigerous cords of the fetal ovary. Its expression is down-regulated as epithelial cells differentiate into granulosa cells during the process of folliculogenesis occurring at birth. If this process is impaired by the lack of oocytes, ovigerous cord organization is maintained together with Lhx9 expression. In conclusion, Lhx9 expression can be inversely correlated with the commitment into a differentiation pathway of the different categories of mesothelium-derived cells of the gonad.


Subject(s)
Cell Differentiation/physiology , Homeodomain Proteins/genetics , Ovary/metabolism , Testis/metabolism , Amino Acid Sequence , Animals , Base Sequence , Chick Embryo , Epithelium/embryology , Epithelium/metabolism , Female , Gene Expression Profiling , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/metabolism , LIM-Homeodomain Proteins , Male , Molecular Sequence Data , Ovary/embryology , Rats , Testis/embryology , Transcription Factors
7.
Dev Dyn ; 222(2): 228-37, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11668600

ABSTRACT

Aromatase inhibitors administered prior to histological signs of gonadal sex differentiation can induce sex reversal of genetic female chickens. Under the effects of Fadrozole (CGS 16949A), a nonsteroidal aromatase inhibitor, the right gonad generally becomes a testis, and the left gonad a testis or an ovotestis. We have compared the expression pattern of the genes encoding AMH (the anti-Müllerian hormone), SF1 (steroidogenic factor 1), and SOX9 (a transcription factor related to SRY) in these sex-reversed gonads with that in control testes and ovaries, using in situ hybridization with riboprobes on gonadal sections. In control males, the three genes are expressed in Sertoli cells of testicular cords; however, only SOX9 is male specific, since as observed previously AMH and SF1 but not SOX9 are expressed in the control female gonads. In addition to testicular-like cords, sex-reversed gonads present many lacunae with a composite, thick and flat epithelium. We show that during embryonic and postnatal development, AMH, SF1 and SOX9 are expressed in the epithelium of testicular-like cords and in the thickened part but not in the flattened part of the epithelium of composite lacunae. AMH and SF1 but not SOX9 are expressed in follicular cells of ovotestes. Coexpression of the three genes, of which SOX9 is a specific Sertoli-cell marker, provides strong evidence for the transdifferentiation of ovarian into testicular epithelium in gonads of female chickens treated with Fadrozole.


Subject(s)
Aromatase Inhibitors , DNA-Binding Proteins/genetics , Disorders of Sex Development , Glycoproteins , Growth Inhibitors/genetics , High Mobility Group Proteins/genetics , Testicular Hormones/genetics , Transcription Factors/genetics , Animals , Anti-Mullerian Hormone , Chick Embryo , Chickens , Enzyme Inhibitors/pharmacology , Fadrozole/pharmacology , Female , Fushi Tarazu Transcription Factors , Gene Expression Regulation, Developmental , Homeodomain Proteins , Male , Ovary/physiology , RNA, Messenger/analysis , Receptors, Cytoplasmic and Nuclear , SOX9 Transcription Factor , Sertoli Cells/physiology , Sex Differentiation/drug effects , Sex Differentiation/physiology , Steroidogenic Factor 1
8.
J Virol ; 75(2): 1048-53, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11134319

ABSTRACT

Porcine xenografts may offer a solution to the shortage of human donor allografts. However, all pigs contain the porcine endogenous retrovirus (PERV), raising concerns regarding the transmission of PERV and the possible development of disease in xenotransplant recipients. We evaluated 11 antiretroviral drugs licensed for human immunodeficiency virus type 1 (HIV-1) therapy for their activities against PERV to assess their potential for clinical use. Fifty and 90% inhibitory concentrations (IC(50)s and IC(90)s, respectively) of five nucleoside reverse transcriptase inhibitors (RTIs) were determined enzymatically for PERV and for wild-type (WT) and RTI-resistant HIV-1 reference isolates. In a comparison of IC(50)s, the susceptibilities of PERV RT to lamivudine, stavudine, didanosine, zalcitabine, and zidovudine were reduced >20-fold, 26-fold, 6-fold, 4-fold, and 3-fold, respectively, compared to those of WT HIV-1. PERV was also resistant to nevirapine. Tissue culture-based, single-round infection assays using replication-competent virus confirmed the relative sensitivity of PERV to zidovudine and its resistance to all other RTIs. A Gag polyprotein-processing inhibition assay was developed and used to assess the activities of protease inhibitors against PERV. No inhibition of PERV protease was seen with saquinavir, ritonavir, indinavir, nelfinavir, or amprenavir at concentrations >200-fold the IC(50)s for WT HIV-1. Thus, following screening of many antiretroviral agents, our findings support only the potential clinical use of zidovudine.


Subject(s)
Antiviral Agents/pharmacology , Endogenous Retroviruses/drug effects , Protease Inhibitors/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Amino Acid Sequence , Animals , Drug Resistance, Microbial , Drug Resistance, Multiple , Endogenous Retroviruses/enzymology , Endogenous Retroviruses/physiology , Endopeptidases/metabolism , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , HIV-1/enzymology , Humans , Microbial Sensitivity Tests/methods , Molecular Sequence Data , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism , Swine , Virus Cultivation
9.
Reprod Fertil Dev ; 13(5-6): 435-41, 2001.
Article in English | MEDLINE | ID: mdl-11833941

ABSTRACT

A previous study in the rat (Pollard et al. 1990) established that caffeine, when administered during pregnancy, significantly inhibited the differentiation of the seminiferous cords and subsequent Leydig cell development in the interstitium. However, that study could not distinguish between the direct effects of caffeine and/or the intermediary secondary toxic effects of metabolites such as theophylline and theobromine. Because the fetus lacks the appropriate enzyme systems, clearance of toxic substances takes place via the placenta and maternal liver. Thus, a suitable in vitro system can effectively differentiate between primary and secondary drug effects. In the present study, 13-day-old fetal testis, at the stage of incipient differentiation, were cultured for 4 days in vitro in the presence of graded doses of caffeine, theophylline or theobromine. It was found that explants exposed to caffeine or theobromine differentiated normally, developing seminiferous cords made up of Sertoli and germ cells, soon followed by the differentiation of functionally active Leydig cells appearing in the newly formed interstitium. However, explants exposed to theophylline failed to develop seminiferous cords and, as a consequence, Leydig cells. In conclusion, insights obtained from different experimental methods, such as organ culture or whole organism studies, are not always identical. It may be prudent, therefore, to take into account that certain experimental techniques, despite providing valuable information, may require confirmation by other test methods in order to obtain an in-depth understanding of mechanisms of action involved.


Subject(s)
Caffeine/pharmacology , Testis/drug effects , Testis/embryology , Theobromine/pharmacology , Theophylline/pharmacology , Animals , Caffeine/toxicity , Cell Differentiation , Female , Gestational Age , Leydig Cells/drug effects , Male , Maternal-Fetal Exchange , Organ Culture Techniques , Pregnancy , Rats , Rats, Wistar , Seminiferous Tubules/drug effects , Seminiferous Tubules/embryology , Theobromine/toxicity , Theophylline/toxicity
11.
J Biol Chem ; 274(2): 770-5, 1999 Jan 08.
Article in English | MEDLINE | ID: mdl-9873014

ABSTRACT

Proliferation and differentiation of progenitor stem cells are mainly controlled by diffusible and adhesion molecules. Stem cell factor (SCF), an essential regulator of spermatogenesis produced by Sertoli cells, utilize both modes of cell to cell communication. Indeed, SCF exists in soluble (SCFs) and membrane-bound (SCFm) forms, which are required for a complete spermatogenesis, and are generated by alternative splicing of optional exon 6, encoding sites of proteolysis. We show that in the mouse testis, the alternative splicing of SCF is developmentally regulated. SCFs predominates in fetal and neonatal gonads and is then replaced by SCFm in the prepubertal and adult gonads. By sequencing SCF exon 6, we show that the flanking intronic sequences perfectly follow the gt-at rule, suggesting that the basal splicing machinery might not be responsible by itself for exon 6 skipping. Moreover, freshly isolated Sertoli cells mainly express SCFm, but a switch to SCFs occurs after 48 h of culture. We found that this change can be prevented by acidification of the culture medium at pH 6.3 or by addition of lactate. The sustained synthesis of SCFm at low pH was no longer observed in the presence of cycloheximide, suggesting that SCF exon 6 skipping requires de novo protein synthesis. Accordingly, UV cross-linking experiments show that nuclear Sertoli cell protein(s) bind in a sequence-specific manner to exon 6. Together, our data allow the proposal of an integrated mechanism in which the synthesis of lactate by Sertoli cells is used in the same time as an energetic substrate for germ cells and as a promoter of their survival/proliferation through the production of SCFm.


Subject(s)
Alternative Splicing , RNA Precursors/genetics , RNA, Messenger/genetics , Stem Cell Factor/genetics , Testis/metabolism , Animals , Base Sequence , Cells, Cultured , DNA , Exons , Gene Expression Regulation, Developmental , Hydrogen-Ion Concentration , Introns , Lactic Acid/metabolism , Male , Mice , Molecular Sequence Data , Spermatogonia/cytology , Spermatogonia/metabolism , Testis/cytology
12.
Differentiation ; 63(5): 273-84, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9810706

ABSTRACT

The acidic keratins K18 and K19 have been shown to display a sex-specific expression during gonadal differentiation in the rat. To extend these findings, we have undertaken a study of the expression of genes encoding for K18 and K19 and their basic partner K8 in the mouse from 10.5 days of gestation until adulthood, using immunofluorescence, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). In the urogenital ridge at 10.5 days of gestation, K18, K19, and K8 are present, in both sexes, in coelomic epithelium in the area of the prospective gonad. At 11 days and 10 h of gestation, they are detected in differentiating gonadal blastema. In male gonads at 11 days and 16 h of gestation the first Sertoli cells differentiate. They are stained for anti-Müllerian hormone by immunofluorescence and appear as dispersed cells throughout the blastema. Progressively, they adhere to each other and form differentiating seminiferous cords. K19 disappears as Sertoli cells differentiate. K18 and K8 continue to be detected in Sertoli cells during fetal life and after birth until 14 days postpartum. In the adult testis, no keratin is observed. In differentiating ovaries, the three keratins are present in somatic cells of the ovigerous cords during fetal life and in primordial follicles differentiating from 1-2 days postpartum. In the course of follicular development, K19 is no longer detected as primordial follicles differentiate into growing follicles. K18 and K18 are present in all stages of follicular development. These results show both differences and similarities with the results previously obtained in the rat. In the mouse, in contrast to the rat, keratins are detected in adult ovaries, and K18 is found in undifferentiated gonads and in ovaries. K18 is, thus, not specific to the testis in the mouse, as it is in the rat. In both species, K19 ceases to be expressed in male gonads as Sertoli cells differentiate and form seminiferous cords. The present observations confirm that downregulation of K19 gene expression in the fetal testis is one of the earliest molecular events attesting the commitment of the undifferentiated gonad to the male differentiative pathway.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Gonads/metabolism , Keratins/genetics , Sex Characteristics , Animals , Cell Differentiation/physiology , Embryonic and Fetal Development/physiology , Female , Fluorescent Antibody Technique , Gestational Age , Gonads/embryology , Gonads/growth & development , In Situ Hybridization , Male , Mice , Ovary/metabolism , Testis/metabolism , Time Factors
13.
J Virol ; 72(12): 9986-91, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9811736

ABSTRACT

Recent interest in the use of porcine organs, tissues, and cells for xenotransplantation to humans has highlighted the need to characterize the properties of pig endogenous retroviruses (PERVs). Analysis of a variety of pig cells allowed us to isolate and identify three classes of infectious type C endogenous retrovirus (PERV-A, PERV-B, and PERV-C) which have distinct env genes but have highly homologous sequences in the rest of the genome. To study the properties of these env genes, expression plasmids for the three env genes were constructed and used to generate retrovirus vectors bearing corresponding Env proteins. Host range analyses by the vector transduction assay showed that PERV-A and PERV-B Envs have wider host ranges, including several human cell lines, compared with PERV-C Env, which infected only two pig cell lines and one human cell line. All PERVs could infect pig cells, indicating that the PERVs have a potential to replicate in pig transplants in immunosuppressed patients. Receptors for PERV-A and PERV-B were present on cells of some other species, including mink, rat, mouse, and dog, suggesting that such species may provide useful model systems to study infection and pathogenicity of PERV. In contrast, no vector transduction was observed on nonhuman primate cell lines, casting doubt on the utility of nonhuman primates as models for PERV zoonosis. Interference studies showed that the three PERV strains use receptors distinct from each other and from a number of other type C mammalian retroviruses.


Subject(s)
Endogenous Retroviruses/classification , Endogenous Retroviruses/pathogenicity , Swine/virology , Animals , Base Sequence , Cell Line , DNA Primers/genetics , Dogs , Endogenous Retroviruses/genetics , Genes, env , Genetic Vectors , Genome, Viral , Humans , Lac Operon , Mice , Rats , Recombination, Genetic , Species Specificity , Transduction, Genetic , Viral Interference
14.
Dev Dyn ; 212(4): 522-32, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9707325

ABSTRACT

In mammals, anti-Müllerian hormone (AMH) is produced by Sertoli cells from the onset of testicular differentiation and by granulosa cells only after birth. SOX9, a transcription factor related to the testis-determining factor SRY, is expressed in mouse testis 1 day before AMH. To determine the relationship between AMH and SOX9 in birds, we cloned the AMH promoter in search of SOX9 response elements, and we compared the expression of AMH and SOX9 in the gonads of chick embryos using in situ hybridization. Potential SOX response elements were found in the AMH promoter; however, AMH is expressed in both sexes at stage 25, 1 day before the first SOX9 transcripts appear in the male gonads. SOX9 is never expressed in the female. These results do not support the hypothesis that SOX9 could trigger the expression of testicular AMH in the chick but does not exclude a later role in testis development.


Subject(s)
Glycoproteins , Gonads/embryology , Gonads/metabolism , Growth Inhibitors/biosynthesis , High Mobility Group Proteins/biosynthesis , Mullerian Ducts/metabolism , Sex Differentiation/physiology , Testicular Hormones/biosynthesis , Testis/enzymology , Testis/metabolism , Transcription Factors/biosynthesis , Animals , Anti-Mullerian Hormone , Base Sequence , Chick Embryo , Chromosome Mapping , Cloning, Molecular , Female , Gonads/chemistry , Growth Inhibitors/analysis , Growth Inhibitors/genetics , Growth Inhibitors/physiology , High Mobility Group Proteins/physiology , In Situ Hybridization, Fluorescence , Male , Molecular Sequence Data , Mullerian Ducts/chemistry , Mullerian Ducts/physiology , Ovary/chemistry , Ovary/enzymology , Ovary/metabolism , Regulatory Sequences, Nucleic Acid/genetics , SOX9 Transcription Factor , Sequence Analysis, DNA , Testicular Hormones/analysis , Testicular Hormones/genetics , Testicular Hormones/physiology , Testis/chemistry , Transcription Factors/physiology
15.
Endocrinology ; 139(4): 2163-70, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9529006

ABSTRACT

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 time-course study with a long-lasting formulation showed that rise in NOS I developed rapidly after a lag of approximately 5 h, with a 2-fold increase detectable after 8 h and a maximal 4.5-fold after 48 h. The level declined afterwards in a manner consistent with homologous desensitization that may occur in the continuous presence of GnRH; however, the profile was different and delayed compared with those of gonadotropin release. As observed for NOS I protein, NOS I messenger RNA concentration was increased by castration or GnRH agonist and reduced by steroids or GnRH antagonist. Taken together, these data demonstrate that steroids indirectly regulate NOS I messenger RNA and protein levels, through the hypothalamic modulation of GnRH, which represents the primary regulator of NOS I. No effect of steroids on NOS I was seen in the posterior lobe. NADPH-diaphorase histochemistry coupled to immuno-identification of the cells revealed that the treatments affecting the concentration of NOS I concomitantly altered the activity but exclusively in gonadotrophs and not in folliculo-stellate cells (which do not respond to GnRH), reinforcing the idea that GnRH played a major regulatory role. Expression in gonadotrophs of a GnRH-dependent NOS I and the ensuing production of nitric oxide represents a potentially novel signaling pathway for the neuropeptide in the anterior pituitary, consistent with the previously reported GnRH-induced cGMP production, the role of which remains to be evaluated.


Subject(s)
Estradiol/pharmacology , Gene Expression/drug effects , Gonadotropin-Releasing Hormone/pharmacology , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Pituitary Gland, Anterior/enzymology , Testosterone/pharmacology , Animals , Blotting, Western , Drug Tolerance , Gonadotropin-Releasing Hormone/agonists , Gonadotropin-Releasing Hormone/antagonists & inhibitors , Histocytochemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Male , NADPH Dehydrogenase/analysis , Orchiectomy , RNA, Messenger/metabolism , Rats , Rats, Wistar , Triptorelin Pamoate/pharmacology
16.
Mech Dev ; 52(2-3): 199-207, 1995 Aug.
Article in English | MEDLINE | ID: mdl-8541209

ABSTRACT

It has been shown previously that acidic K18 and K19 keratins display a differential immunohistochemical pattern of expression during sexual differentiation of the gonads in the rat (Fridmacher et al. (1992) Development 115, 503-517). The present results indicate that K18 and K19 gene expression is regulated at the transcriptional level. The analysis was performed by Northern Blot, reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization. PCR products were cloned, sequenced and used as species-specific K18 and K19 riboprobes for in situ hybridization. K19 mRNA but not K18 mRNA was detected in undifferentiated gonads and in somatic cells of ovarian cords throughout the fetal ovary development. K18 mRNA expression appeared in male gonads, at 13.5 days of gestation, at the onset of testicular differentiation, as the first Sertoli cells differentiated and aggregated to form seminiferous cords. As testicular differentiation progressed, K19 mRNA disappeared and, from 14.5 days of gestation on, fetal Sertoli cells expressed exclusively K18 mRNA. The changes in the transcriptional activity of K19 and K18 genes, observed in male gonads, occur characteristically at the very beginning of testicular differentiation. In the male pathway of sexual differentiation, the switch in K19/K18 gene expression is, in addition to the activation of the anti-Müllerian hormone gene, the most precocious regulative event occurring after the expression of the testis determining factor SRY.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Keratins/genetics , Sex Differentiation/genetics , Testis/embryology , Animals , Base Sequence , Female , Gestational Age , Male , Molecular Sequence Data , Ovary/embryology , RNA, Messenger/biosynthesis , Rats , Rats, Wistar
17.
Contracept Fertil Sex ; 22(9): 535-8, 1994 Sep.
Article in French | MEDLINE | ID: mdl-7994314

ABSTRACT

Morphological differentiation of gonads results within successive morphogenetic steps beginning with the formation of the undifferentiated gonad, identical in both sexes, through the differentiation of the characteristic gonadal structures, seminiferous tubes in the testis and follicles in the ovary. In this paper, we report recent data on each of these steps and particularly on the origin of somatic gonadal cells. Results from our laboratory are summarized with special emphasis on the relationships between cellular differentiation and morphogenetic processes.


Subject(s)
Gonads/growth & development , Sex Differentiation/physiology , Animals , Gonads/embryology , Gonads/physiology , Humans , Rats
18.
Development ; 115(2): 503-17, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1385062

ABSTRACT

The expression of cytokeratins (CKs) 8, 18 and 19 was analyzed in male and female rat gonads from the undifferentiated stage (12.5 days of gestation) until two weeks after birth by indirect immunofluorescence, using specific monoclonal antibodies anti-CK 8 (LE41), anti-CK 19 (LP2K) and anti-CK 18 (LE65 and RGE53). In the undifferentiated blastema, the somatic cells were stained for CK 8 and CK 19, whereas no detectable immunoreactivity for CK 18 was obtained. The same staining CK pattern was observed in ovaries, in the somatic cells of ovigerous cords and in primary follicles. The staining was progressively decreasing in growing follicles after one week after birth. At the onset of testicular differentiation, when the first Sertoli cells differentiate in the gonad of 13.5-day old male fetuses, positive staining for CK 18 became evident, in addition to CK 8 and CK 19 expression. In the following days, CK 8, CK 18 and CK 19 were detected in Sertoli cells in the differentiating seminiferous cords, but progressively the reactivity for CK 19 decreased and was no longer observed after 18.5-19.5 days of gestation. In all cases, CKs were found to be coexpressed with vimentin, and germ cells were negative for both vimentin and CKs. The results reported here show first, that CKs are expressed before sexual differentiation in gonadal blastema in which no epithelial organization is observed, and second, that there is a CK 18/CK 19 shift in expression during morphogenesis of the testis which is not observed in the differentiating ovary. Future studies will have to determine whether these differences in CK expression are due to epitope-masking phenomena or to the regulation of CK synthesis.


Subject(s)
Genes/genetics , Keratins/genetics , Ovary/embryology , Sex Differentiation/genetics , Testis/embryology , Animals , Electrophoresis, Gel, Two-Dimensional/methods , Female , Fluorescent Antibody Technique , Gene Expression , Immunoblotting/methods , Immunohistochemistry , Keratins/analysis , Male , Microscopy, Electron , Ovary/chemistry , Ovary/ultrastructure , Rats , Rats, Wistar , Testis/chemistry , Testis/ultrastructure
19.
J Electron Microsc Tech ; 19(2): 172-88, 1991 Oct.
Article in English | MEDLINE | ID: mdl-1748901

ABSTRACT

The fetal testis is not merely a precursor of the adult organ: it is indeed an endocrine gland whose function is the masculinization of the fetus. It differs physiologically and morphologically from the adult testis. In this paper, the first stages of testicular differentiation in the rat are described, with special emphasis on the ultrastructural aspects. At the stage of 13.5 days after fertilization, the first Sertoli cells differentiate; they are characterized by a voluminous and little electron dense cytoplasm, a well-developed RER formed by vesicles and short cisternae filled with a flocculent material. Progressively, they polarize and adhere to one another by adherens-like junctions and cytoplasmic interdigitations to form the differentiating seminiferous cords. In the basal part of the Sertoli cells, a mat of microfilaments differentiates under the plasmalemma, while cytoplasmic blebs protruding in the extracellular space tend to disappear. A continuous basal lamina delineating the seminiferous cords begins to appear on day 14.5 and becomes widespread on day 15.5. These observations, when compared with other data from the literature, emphasize the fact that the differentiation of the Sertoli cells is the first morphological event during testicular differentiation. A possible role of the Sertoli cells in the subsequent organogenesis of the testis is suggested.


Subject(s)
Fetus/ultrastructure , Sertoli Cells/ultrastructure , Testis/embryology , Animals , Cell Differentiation , Female , Gonads/ultrastructure , Male , Morphogenesis , Rats , Seminiferous Tubules/ultrastructure , Testis/ultrastructure
20.
Bull Assoc Anat (Nancy) ; 75(228): 15-20, 1991 Mar.
Article in French | MEDLINE | ID: mdl-1782454

ABSTRACT

Testicular morphogenesis in the rat foetus, requires at least two distinct steps of differentiation. One of these steps is the differentiation of Sertoli cells. This is the first morphological event which allows identification of the male gonad. The other truly morphogenetic step is the acquisition of epithelial characteristics by Sertoli cells: aggregation, polarization, expression of cytokeratin. This step, which is obtained in vitro if gonads are cultured in synthetic medium, is impaired when gonads are cultured in serum-supplemented medium. In both cases Sertoli cells differentiate morphologically and physiologically. Immunohistochemically detectable cytokeratin (monoclonal antibody Lu-5) was found in Sertoli cells incorporated in seminiferous cords either in vivo or in vitro, but not in undifferentiated somatic cells or in Sertoli cells of cordless gonads. These results suggest that the expression of cytokeratin, in the differentiating testis, cannot be considered as a marker of the origin of the cells, but rather is related to the adhesion of Sertoli cells to each other.


Subject(s)
Testis/cytology , Cell Differentiation , Epithelial Cells , Humans , Male , Microscopy, Electron , Morphogenesis , Testis/embryology , Testis/ultrastructure
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