The effects of an in utero exposure to 2,3,7,8-tetrachloro-dibenzo-p-dioxin on male reproductive function: identification of Ccl5 as a potential marker.

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.

Fibroblast growth factor (FGF) 2 and FGF9 mediate mesenchymal-epithelial interactions of peritubular and Sertoli cells in the rat testis.

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.

Switch in the expression of the K19/K18 keratin genes as a very early evidence of testicular differentiation in the rat.

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.

The potential hazards of xenotransplantation: an overview.

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.

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 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.

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 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.

Lhx9 expression during gonadal morphogenesis as related to the state of cell differentiation.

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.

Maternal and foetal corticosterone levels during late pregnancy in rats.

Adrenal and plasma corticosterone levels were determined in rat foetuses and in intact or adrenalectomized mothers during late pregnancy. Foetal adrenal and plasma corticosterone concentrations reached a peak on day 19 of pregnancy, while maternal plasma corticosterone increased on day 18 and remained high until parturition. From day 18, mothers adrenalectomized on day 14 had corticosterone levels similar to those of intact pregnant rats. At every stage of gestation (except day 21) plasma corticosterone levels were higher in the foetuses than in the mothers. The corticosterone concentration in the maternal plasma correlated with the number of live foetuses during the last 3 days of gestation. These results suggest that corticosterone can cross the placenta from foetus to mother as early as day 18 and that the foetus contributes to the maternal corticosterone pool after day 18.

Dissociation between testicular morphogenesis and functional differentiation of Leydig cells.

The aim of the study was to determine whether Leydig cells differentiate in vitro in gonads in which the formation of seminiferous cords is prevented by culture in a medium containing fetal calf serum. Appearance of 3 beta-hydroxysteroid dehydrogenase-positive cells and release of testosterone in the medium occurred at the same age irrespective of whether or not the gonads developed seminiferous cords. It is concluded therefore that testicular morphogenesis with the formation of seminiferous cords is not a prerequisite for the emergence and functional differentiation of Leydig cells.

Effects of caffeine and its reactive metabolites theophylline and theobromine on the differentiating testis.

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.

Critical study of endogenous peroxidase activity: its role in the morphofunctional setting of the thyroid follicle in the rat fetus.

Endogenous peroxidase is associated with thyroglobulin iodination. It first appears at 17 day of pregnancy in rat fetal thyroid and seems to be synthesized within the perinuclear cistern and the rough endoplasmic reticulum. Then, the enzyme is transferred by vesicles up to the follicular lumen where thyroglobulin iodination takes place. The different steps of this process can occur within a very short time, since peroxidase activity is already located in the apical plasma membrane of follicles formed as early as 17 days of pregnancy, which is consistent with autoradiographic data showing iodinated thyroglobulin at 17 days. It can be concluded that the follicle is functional as soon as it is formed.

[Sertoli cells and organogenesis of the fetal testis (author's transl)]. / Cellules de Sertoli et organogenèse du testicule foetal.

In previous papers (1, 2), it was shown that in the rat fetus, testicular differentiation begins 13 days after fertilization, in the depth of the primordium near to mesonephric tubules. A few cells swell and differentiate into Sertoli cells which encompass the germ cells. In the meantime they delineate locally one part of the contour of the developing seminiferous cord, which is completed by the recruitment of new cells during the next hours. Under the electron microscope the differentiation of the Sertoli cells involves cytoplasmic and organelles changes, typical junctions and microfilaments toward the external surface of the seminiferous cord. Testicular differentiation can be obtained in vitro in cultures made in a synthetic medium without or with addition of chick embryo extract. Fetal calf serum prevents the organogenesis of the sex cords or produces the disintegration of those already differentiation (on day 14). These data clearly underline some of the cellular processes involved in the differentiation of the seminiferous cords.

[Initial stages of gonadal differentiation]. / Stades initiaux de la différenciation gonadique.

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.

[Reduction of germ cell number induced in vitro by the testis in fetal rat ovary may be caused by factors other than anti-Müllerian hormone]. / La réduction du nombre de cellules germinales induite in vitro par le testicule dans l'ovaire foetal de rat pourrait être due à un facteur distinct de l'hormone anti-Müllérienne.

Fetal testes explanted at 16.5 days and cultured with female genital tracts from 13.5-day-old rat fetuses strongly inhibited the Müllerian ducts and reduced the number of ovarian germ cells. Such a reduction was not obtained during cultures with testes from 13.5 days, even though they clearly inhibited Müllerian ducts. When testes from 16.5 days were cultured at distance from the female tracts only the loss of germ cells was observed. These results suggest that testes from 16.5 days produce a diffusible factor distinct from AMH and which reduces the number of germ cells in cultured ovaries.

Control mechanisms of testicular differentiation.

In this paper the importance of unknown factors responsible for the initial differentiation of a gonadal primordium is stressed. The hypothesis that in the absence of testis determining genes (TDG) the indifferent gonad is programmed to become an ovary is considered further. The TDG(s) are expressed only among cells already marked as gonadal cells, and they seem mainly to change the chronological sequence and intensity of expression of processes common to both sexes. The chronology of the normal events necessary for testicular differentiation and the fact that some of these events can be dissociated from one another under experimental conditions in vitro, suggest that many genes are involved in testicular differentiation and that the so-called testis-determining genes are probably regulatory genes.

Expression of fibronectin and laminin in fetal male gonads in vivo and in vitro with and without testicular morphogenesis.

Sertoli cell differentiation occurs in vitro, even when testicular morphogenesis is inhibited by addition of serum to the culture medium (Magre, S. and A. Jost: Proc. Natl. Acad. Sci. USA 81, 7831-7834 (1984]. Using indirect immunohistochemical technique, we have studied the expression of fibronectin and laminin in gonads lacking testicular morphogenesis, as compared to in vivo controls and gonads cultured in synthetic medium. In undifferentiated gonads in vivo, fibronectin and laminin are distributed uniformly in the blastema. If testicular differentiation occurs in vivo, laminin is detected only in the basement membranes; when it occurs in vitro, laminin is found both in the basement membranes and among the stromal tissue. In gonads without seminiferous cords (cultured in serum-supplemented medium), fibronectin and laminin are both present, they are uniformly distributed among the gonadal cells.

The initial phases of testicular organogenesis in the rat. An electron microscopy study.

The paper is devoted to the study of the very first stages of testicular organogenesis in the rat, i.e. to the study of the initial stages of formation of the seminiferous cords. It takes over and extends the results of a previous histological study (Jost, 1972). The tissues were fixed with glutaraldehyde or glutaraldehyde-paraformaldehyde and post-fixed with osmium tetroxide. Examination with the optical microscope of semi-thick sections confirms that the differentiation begins at the stage of 13 days after fertilization; an increasing number of primitive Sertoli cells appear, first in the depth of the gonad, join each other and form the seminiferous cords. Study with the electron microscope reveals three characteristic criteria of the early differentiation of the Sertoli cells, namely : 1) an abundant cytoplasm, only slightly dense to electrons and containing short laminae or vesicles of rough endoplasmic reticulum; 2) complex contact zones involving infoldings and interdigitations of the membranes; 3) a layer of microfilaments beneath the flattened surface of the cells, that limits the outer surface of the seminiferous cords. These differentiation processes taking place at the stage of 13 days, are described.

Dissociation between testicular organogenesis and endocrine cytodifferentiation of Sertoli cells.

Differentiation of the rat testis from the undifferentiated primordium begins with the appearance of a new cell type characterized by a large and clear cytoplasm. These cells aggregate, enclose germ cells, and progressively form seminiferous cords. Therefore, they were considered primordial Sertoli cells. A similar process was obtained in vitro in explants cultured in a synthetic medium. On the contrary, when fetal calf serum was added to the medium, the organization of seminiferous cords was impaired; large clear cells appeared, but they did not aggregate. Instead, they remained scattered throughout the abnormal gonad. The present experiments were undertaken to verify whether these cells are in fact Sertoli cells. The production of Müllerian inhibitor is a marker of fetal Sertoli cells. Therefore, undifferentiated gonadal primordia from 12-day 16-hr old male rat fetuses were cultured for 2 days in vitro with serum and then associated for 3 days with 14.5-day-old sex ducts from female fetuses. Müllerian ducts were inhibited as well by the abnormal cordless gonads as by those with differentiated sex cords. These experiments confirm previous views on testicular development and demonstrate that differentiation of Sertoli cells may take place quite independently of the testicular cord formation.