Automatic correction of rotating ultrasound bio microscopy acquisitions for the segmentation of the eye anterior segment.

We have developed a rotating 3D probe prototype in order to acquire the anterior segment of the eye in three dimensions. The acquisition accuracy has to be sufficient to allow for the use of automatic segmentation of the provided data, and thus generate a 3D structure of the eye, for which it could be easier to obtain measurements than in 2D images. We have created an image post processing scheme in order to compensate for vibrations and eye movements during acquisition that are associated with increased noise. These tools have been applied to 92 volume datasets acquired on 21 patients in pre-operative conditions. Acquisition noise was reduced by 97% in specific conditions with respect to data acquired without correction.

[Long-term results of posterior chamber phakic intraocular lens implantation for correction of high ametropia]. / Résultats à long terme de l'implantation phaque de chambre postérieure pour la correction des amétropies fortes.

PURPOSE: To assess efficacy, stability and safety of posterior chamber phakic intraocular lens implantation with STAAR Visian ICL for correction of high ametropia, with a mean follow-up of 5 years (3.5-10 years). PATIENTS AND METHODS: Ninety eyes of 53 highly ametropic patients (45 myopia, ten hyperopia and 35 with mixed astigmatism) were included in a retrospective single-surgeon study, using primarily the V4 ICL model (87 eyes). We studied pre- and postoperative refractive efficacy, endothelial cell density, crystalline lens opacification and intraocular clearances within the various compartments of the eye. RESULTS: Mean uncorrected visual acuity was 0.77 at the 12th postoperative month; 17 of 90 eyes required adjunctive photoablation for residual astigmatism. Forty-eight percent of eyes gained at least one line of best corrected visual acuity. After implantation, the decrease in endothelial cell density remained stable at 0.69%/year, and 91% of eyes showed no opacification of the crystalline lens. Mean endothelium-ICL and ICL-crystalline lens distances were 2.41 mm and 0.52 mm respectively. Overall patient satisfaction achieved was 96% at 36 months postoperatively. DISCUSSION AND CONCLUSION: These results demonstrate efficacy, stability and safety of the ICL V4 phakic IOL for the correction of high ametropia. Long-term follow-up did not show a significant increase in cataract formation in implanted eyes.

[Endogenous bacterial endophthalmitis related to dental abscess: case report]. / Endophtalmie endogène bactérienne sur abcès dentaire: à propos d'un cas.

INTRODUCTION: Endogenous bacterial endophthalmitis (EBE) is an intraocular bacterial infection transmitted via a hemotogenous route, usually occuring in immunocompromised patients with bacteremia spreading from extraocular foci of infection. We report a case of EBE secondary to a dental infection occurring in an immunocompetent patient. CASE REPORT: A 61-year-old-man with no past medical history other than a dental bridge on the 13th tooth was referred for rapid onset hypertensive fibrinous panuveitis of the right eye. He presented with profound visual loss in this eye, an absent pupillary reflex and an obscured fundus. The left eye was normal. Neither fever nor constitutional symptoms were noted at presentation. Work-up revealed a mild inflammatory syndrome with increased C-reactive protein, a high antistreptolysin O titer, and an abscess of the dental bridge. The clinical picture worsened rapidly, suggesting the possibility of EBE secondary to an organism of dental origin. Aqueous humor polymerase chain reaction (PCR) was positive for streptococci, which could not be identified more specifically. Removal of the dental bridge in combination with systemic and intravitreal multiple antiobiotic therapy achieved a rapid cure of the EBE. Vitrectomy combined with phacoemulsification was performed later in order to clear media opacities. At 6 months follow-up, best-corrected visual acuity was 8/10 with no recurrent inflammation. CONCLUSION: This case shows that EBE can occur in the absence of predisposing factors but with an extraocular infectious focus as simple as a dental abscess. In the absence of associated septicemia, with the involved tooth superior and ipsilateral to the affected eye, a spread of the infection by retrograde venous flow should be suspected. The prognosis for visual function may be good if early diagnosis and immediate treatment can be provided.

In vivo and in vitro effects of prochloraz and nonylphenol ethoxylates on trout spermatogenesis.

We investigated the effects of in vivo exposure to non-lethal concentrations of two chemicals commonly discharged into the aquatic environment, prochloraz and nonylphenol diethoxylate (NP2EO - Igepal(R) 210), on the development of spermatogenesis in trout. The in vitro effects on basal and insulin-like growth factor-1 (IGF-I) stimulated DNA synthesis by early germ cells were also studied. In vivo, rainbow trout were exposed for 2 or 3 weeks to waterborne prochloraz (21 and 175 nmol/l) and/or NP2EO (68-970 nmol/l) renewed continuously, or periodically. Only the highest concentrations of NP2EO (225-970 nmol/l) induced a significant increase in blood plasma vitellogenin in juvenile or maturing male trout. When prepubertal fish were exposed for 15 days to prochloraz, the spermatogenetic process was significantly inhibited as shown by the stage of gonadal development reached 3 weeks after exposure. This effect was, to a great extent, reversible within 9 weeks post-exposure. When fish in the initial stage of spermatogenesis were exposed for 21-27 days to 580 nmol/l NP2EO, a 20-40% reduction of the gonadosomatic index was observed 4.5 weeks post-exposure, and the spermatogenetic process was partly inhibited. In vitro, testicular cells obtained at different stages of spermatogenesis were cultured for 4.5 days in the presence or not of the tested molecules and with IGF-I or not. 3H-thymidine (3H-Tdr) incorporation was measured according to Loir (Mol. Reprod. Dev. 53 (1999) 424) and 125I-IGF-I specific binding was determined according to Le Gac et al. (Mol. Reprod. Dev. 44 (1996) 35). Irrespective of the spermatogenetic stage, basal 3H-Tdr incorporation was decreased by prochloraz concentrations > or =10 micromol/l. The presence of IGF-I (10-100 ng/ml) stimulated 3H-Tdr incorporation; this response to IGF-I began to decrease at 25-50 micromol/l prochloraz. In parallel, a dose-dependent increase of IGF-I specific binding was induced by prochloraz 1-100 micromol/l. Similarly, basal and IGF-I-stimulated 3H-Tdr incorporation was decreased by nonylphenol polyethoxylate (NpnEO; starting at 10 micromol/l), NP2EO and NP (30 micromol/l); a dose-dependent increase of IGF-I specific binding was also induced by NP and NPnEO. While 1-100 nmol/l 17beta-estradiol had no effect in our in vitro system, Triton(R) X-100 acted as NPnEO on 3H-Tdr incorporation. Beside their known endocrine disrupting effects on sex steroid production or action, these lipophilic molecules could act on germ cells by disrupting cell membrane receptivity to peptide hormones like growth factors.

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation.

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.

Spermatogonia of rainbow trout: I. Morphological characterization, mitotic activity, and survival in primary cultures of testicular cells.

Prerequisites of developing in vitro studies for a better understanding of the control mechanisms underlying the proliferation and differentiation of spermatogonia (Go) in the teleost testis are: (1) to be able to identify the different types of Go; (2) to maintain in culture the structural relationships occurring in situ between the various testicular cell types, as intact as possible; and (3) to know how the Go survive and proliferate in culture for several days. After very gentle homogenization of trout testes treated with collagenase, a cell suspension containing mainly spermatocysts (one or several Sertoli cells enclosing one Go or a clone of germ cells) and clusters of myoid cells and Leydig cells was seeded in culture onto a laminin plus fibronectin coating. After 4.5-6 days in culture, then staining with May-Grünwald and Giemsa reagents, the determination of the nuclear and cellular size of the various Go and of the number of Go present in clones has enabled the identification of two types of large Go, in pairs or alone (Go A) and six successive types of smaller Go (Go B). Cell viability determination by staining with Rhodamine 123/propidium iodide (PI)/Hoechst 33342 and with FITC-Annexin V/PI indicated that after 5-7 days in culture, all the somatic cells and most of the Go were viable. Only some of the Go, mainly among the most differentiated ones, underwent apoptosis, as it was the case for a number of spermatocytes and spermatids increasing with the time in culture. Brdu labelling and 3H-Thymidine (3H-Tdr) incorporation indicated that the proliferative activity of Go was at a maximum after 4.5 days in culture and that the response to at least two molecules (QAYL-IGF-I and GTH-I) remained unchanged between 3 and 6 days. As only very scarce somatic cells from immature/spermatogenetic testes synthesized DNA up to 6 days in culture, the measurement of 3H-Tdr incorporation by cells from such testes reliably reflected synthesis of DNA by only the Go (and eventually also by primary spermatocytes when they are present). In conclusion, this study provides information allowing a detailed analysis of the events related with the mitotic phase of spermatogenesis in the trout and it establishes that primary cultures of testicular cells carried out in the reported conditions represent a useful tool to develop an analysis of the mechanisms participating in the control of this phase.

Spermatogonia of rainbow trout: II. in vitro study of the influence of pituitary hormones, growth factors and steroids on mitotic activity.

At the present time, in spite of recent advances, knowledge about the factors regulating germ cell proliferation in the teleost testis is limited. This study was designed to investigate, in vitro, the ability of various hormones, growth factors, and steroids to influence the proliferation of trout spermatogonia (Go) present in mixed cultures of somatic and germ cells prepared from testes, either prespermatogenetic or spermatogenetic. The tested molecules were usually present for the duration of culture (4.5 days) and 3H-thymidine (3H-Tdr) for the last day in culture. In our cell culture conditions, homologous gonadotropin I (tGTH-I) and growth hormone (tGH) moderately stimulated 3H-Tdr incorporation by Go, with ED50 equal to 5.5 +/- 3.0 and 1.8 +/- 0.4 ng/ml respectively. Insulin growth factor I (rhIGF-I) and fibroblast growth factor (rhFGF-2) stimulated 3H-Tdr incorporation by Go from spermatogenetic testes only, with ED50 equal to 16.2 +/- 9.3 and 2.4 +/- 0.3 ng/ml respectively. The effects of the most efficient concentrations of rhIGF-I combined with those of either tGTH-I or tGH were additive. Seventy to one hundred microM suramin stimulated 3H-Tdr incorporation by Go from testes at all maturation stages and this effect was additive with that of tGTH-I. We assume that this effect of suramin could result from the inhibition of an unidentified antimitogenic factor. No effect was observed with homologous prolactin, human epidermal growth factor, activin A and B, transforming growth factor-beta1, testosterone, 11-ketotestosterone, 17beta-estradiol, pregnenolone, 11beta-hydroxyprogesterone, and 22-hydroxycholesterol. In conclusion, our in vitro results suggest that GTH-I, GH, IGF-I, and FGF-2, are potent in situ modulators of the proliferative activity of trout Go at the time of induction, speeding up, then slowing down spermatogenesis, through direct or indirect additive and/or antagonistic influences.

Spermatogonia of rainbow trout: III. In vitro study of the proliferative response to extracellular ATP and adenosine.

Unlike in higher vertebrates, in fish it is not known whether the nerve supply of the testis can influence testicular functions or not. In addition to neurotransmitters, nerve terminals may release ATP and adenosine in the extracellular medium. On the assumption that these molecules might be released by fibers innervating the teleost testis, it is possible that they participate in the control of testicular function and, maybe, in the control of spermatogonia (Go) proliferation. This study addresses this issue. We have investigated the ability for extracellular ATP and adenosine to influence the in vitro incorporation, either basal or GTH-, IGF-I- and suramin-stimulated, of 3H-thymidine (3H-Tdr) by trout Go. Mixed suspensions of somatic and germ cells prepared from testes, which were immature or spermatogenetic, were cultured usually for 4.5 days in the presence or not of the tested molecules; 3H-Tdr was added during the last day in culture. In our cell culture conditions, 25 to 250 microM adenosine, ATP, ADP, and AMP stimulated the 3H-Tdr incorporation by Go from prespermatogenetic testes and from testes starting spermatogenesis, in a dose-dependent way. The effect of these molecules decreased when the testes were more advanced in spermatogenesis and it became inhibiting when the testes were in mid-spermatogenesis. Five'-N-ethylcarboxamidoadenosine (NECA) was as potent as adenosine in stimulating or inhibiting 3H-Tdr incorporation, while R-N6-(2-phenylisopropyl)adenosine (R-PIA) always had a marked inhibiting effect. Adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS; 25-200 microM), a non-hydrolysable analogue of ATP, which had no effect on Go from prespermatogenetic testes (collected October-February) and from testes in advanced spermatogenesis, stimulated 3H-Tdr incorporation by Go from testes at the beginning of spermatogenesis very efficiently. The order of potency of the different ATP analogues was as follows: ATPgammaS > ATP congruent with alpha,beta-methylene-ATP > UTP > 2-methylthio-ATP. These data suggest that A2 adenosine receptors and P2 receptors would be present on unidentified testicular cells. The stimulating effect of adenosine/ATP was additive with that of either GTH-I or IGF-I or suramin when the cells were from testes at the beginning of spermatogenesis, but adenosine suppressed their effect when the cells were from testes in mid-spermatogenesis. In conclusion, our results suggest that in the trout extracellular adenosine and ATP are able to influence the in vitro proliferation of Go, and are potential candidates for mediating the possible influence of the nervous system on the induction, speeding up, then slowing down of spermatogenesis.

The use of phosphocreatine plus ADP as energy source for motility of membrane-deprived trout spermatozoa.

Live trout spermatozoa initiate flagellar motility for a short period of time (30 s at 18 degrees C), during which their mean beat frequency (BF) decreases steadily from 60 to 20 Hz; motility then stops abruptly. When demembranated, the motility of axonemes lasts much longer, up to 20 min, with high beat frequency, provided that ATP (millimolar concentration) and cAMP (micromolar) are added. In the present paper, the motility of demembranated trout sperm was investigated in the absence of added ATP in various incubation conditions relative to other substrates. Without the addition of exogenous creatine kinase, the addition of phosphocreatine (PCr) and ADP shows the appearance of a progressive activation of all sperm models with BF increasing with time up to high values. Without the addition of cAMP, the BF increases to lower values but flagella propagated poorly coordinated waves for only a few min. Similar progressive activation is also observed when only ADP is added (without any previous in vivo activation) and BF increases up to moderate values. In this latter case, no activation occurs without addition of cAMP. The respective roles of creatine kinase and adenylate kinase in this process were investigated by addition of specific inhibitors such as fluorodinitrobenzene and P1,P5-di(adenosine-5')pentaphosphate in the above described conditions. We conclude from these observations that all the elements necessary for a coupling between ADP/PCr/creatine kinase on one hand and ATP/ADP/dynein on the other appear to be present in trout spermatozoa: thus the existence of a shuttle sustaining this coupling is strongly suggested.

Growth hormone receptors in testis and liver during the spermatogenetic cycle in rainbow trout (Oncorhynchus mykiss).

Changes in growth hormone (GH) receptivity in the testis during the reproductive cycle were studied in rainbow trout (Oncorhynchus mykiss). This necessitated setting up a method to characterize GH receptors (GH-R) in crude testis homogenate. Binding of radiolabeled recombinant rainbow trout GH (125I-rtGH) to crude testis preparation was dependent on testicular tissue concentration. The sites were specific to GH, with no affinity for prolactins, gonadotropins, or insulin. Similar high affinities for 125I-rtGH were obtained with crude testis (4.5 x 10(9) + 0.25 M(-1)) and crude liver preparations (5.1 x 10(9) +/- 0.06 M(-1)) at all stages of spermatogenesis, as well as with testicular membrane preparations (2.4 x 10(9) M(-1)). GH-R concentration in testis was high during the immature stage (590 fmol/g testis); it then decreased regularly from the beginning (stage II, spermatogonia proliferation: 706 fmol/g testis) to the end of spermatogenesis (stage VII: 158 fmol/g testis). Conversely, the absolute testicular capacity increased dramatically between stages IV and VI (1.2-16.2 pmol/2 gonads), when testicular growth is extremely rapid. GH-R concentration in the liver was highest in early stages (6.5-6.7 pmol/g liver); this value then significantly decreased during midspermatogenesis (stage VI: 3.5 pmol/g liver) and returned to immature levels at the end of the cycle. No significant correlation was found between GH-R in testis, GH-R in liver, and plasma GH concentration. Preliminary experiments performed on isolated populations of testicular cells revealed that binding of 125I-rtGH was detectable in Sertoli cell-enriched fractions but not in germ cell populations. We conclude that GH-R are present in the testis during the entire reproductive cycle in rainbow trout, probably in somatic cells and in particular in Sertoli cells.

Creatine kinase in trout male germ cells: purification, gene expression, and localization in the testis.

High creatine kinase (CK) activity (16.5 +/- 7.6 IU/mg) is present in trout spermatozoa. In order to partly characterize the CK isozyme predominantly present in sperm and to study the expression of this protein in spermatogenesis, we purified to homogeneity a CK (s-CK) from trout sperm, by nitrogen cavitation followed by two chromatography steps (DEAE-Trisacryl and Blue Sepharose). Specific antisera to 5-CK were developed. A cDNA encoding for a CK named TCK1, and whose transcript shows enhanced testicular expression, was previously isolated from trout testis (Garber et al., 1990: Biochim Biophys Acta 1087:256-258). A CK subunit expressed in vitro by this cDNA cross-reacts with anti-s-CK. A 21-amino-acid residue sequence near the N-terminus of s-CK is identical to the cDNA-derived sequence of TCK1, which is unlike any previously reported CK sequence. Using in situ hybridization, the TCK1 mRNA was detectable in primary and secondary spermatocytes and in early spermatids. Immunohistochemical staining of testis and various organs revealed that s-CK was confined to testis and, in this organ, to late spermatids and spermatozoa. In gill, some cells exhibited a positive signal, but another study rules out the presence of s-CK in this organ (Garber et al., 1990: Biochim Biophys Acta 1087:256-258). These results demonstrate that s-CK/TCK1 is a germ cell-specific protein, the transcription of which starts in meiotic germ cells, while translation starts in late spermatids.

Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

Few data exist concerning the occurrence and potential role of an insulin-like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF-I cDNA, we confirmed that IGF-I transcription occurs in trout testis. Testicular IGF-I mRNA abundance may be increased by long-term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH-II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF-I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 x 10(10) M-1; Bmax = 10 to 20 fmol/10(7) cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF-I > IGF-II >>> insulin) and the molecular size of its alpha-subunit labelled with 125I-IGF-I (M(r)125-140 kDa). 125I-IGF-II also bound to the type 1 receptor whereas IGF-II/ mannose 6 phosphate receptors could not be detected. Separation of isolated testicular cells by Percoll gradient and centrifugal elutriation provided populations enriched in different types of intratubular cells. IGF-I mRNA (detected by reverse transcription + polymerase chain reaction [PCR]) and IGF-I receptors (measured by competitive binding) were observed to a greater extent in Sertoli cell-enriched populations and in spermatogonia with primary spermatocytes. Therefore, IGF-I is a potential paracrine/autocrine regulator inside the spermatogenic compartment and appears as a possible mediator of GH action at the gonadal level in fish.

20 beta-hydroxysteroid dehydrogenase activity in nonflagellated germ cells of rainbow trout testis.

Nonflagellated germ cells were isolated from rainbow trout testis to determine their ability to synthesize 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta OHP), a progestin involved in the control of the release of sperm. Germ cells were obtained by enzymatic dissociation (collagenase; 3 mg.ml-1, 4.5 h, 12 degrees C) from testes that were immature and at the beginning of spermatogenesis. Somatic cells were eliminated by adhesion to the culture plates. Dose-related amounts of 17,20 beta OHP were measured by RIA in culture media of germ cells incubated with increasing dosages of 17-hydroxyprogesterone (17OHP; 0.05-10 micrograms.ml-1) for 20 h at 12 degrees C. Furthermore, 3H-17,20 beta OHP was identified by chromatography and co-crystallization with a reference in incubating cells provided by 3H-17OHP (2.5 and 4 h, 12 degrees C). Other metabolites were detected but not identified. 11-Ketotestosterone (11KT) was either nondetectable by RIA in control cultures or, when detected, was found at very low levels. In no case was 11KT stimulated by addition of 17OHP or gonadotropin II (GtH II; 400 ng.ml-1); this indicated the absence of contamination by Leydig cells. Thus, to our knowledge, this report demonstrates steroidogenic activities in nonflagellated germ cells of fish testis for the first time. 20 beta-Hydroxysteroid dehydrogenase (20 beta HSD) activity was identified, showing that germ cells are able to synthesize 17,20 beta OHP at an early stage in rainbow trout testis.

Cell-cell interactions in the testis of teleosts and elasmobranchs.

In this paper we present the state of knowledge on cell-cell interactions in the testis of two groups of anamniote vertebrates--teleosts and elasmobranchs--which include most fish. In these fish, the structural organization of the testis differs fundamentally from that which characterizes amniotes in which the germinal tissue is located in tubules open at both ends and consists of a permanent population of Sertoli cells associated with successive stages of germ cell development. In fish, the spermatogenic unit of testis is the spermatocyst, which corresponds to one germ cell or to a clone of isogenetic germ cells, enclosed by one or several Sertoli cells, which form the wall of the cyst. In fish testis, the Sertoli cells do not represent a permanent population of cells. Although both are of the cystic type, the teleost and elasmobranch testes are differently organized. In elasmobranchs, primary spermatogonia and Sertoli cells lie initially free within the interstitial tissue, before becoming sequestered by a basement membrane; the testis is then composed of a mass of spermatocysts which contain many Sertoli cells, each being associated with a clone of germ cells. In contrast, in teleosts, the cysts are confined to large elongated structures limited by a basement membrane. These structures are either lobules originating under the albuginea or tubules which, in contrast to those of mammals, are anastomosed. In the lobules, the spermatocysts start to develop at the blind end of the lobules and migrate towards the efferent system, whereas in the tubules, the spermatocysts are located against the basement membrane, all along the tubules and do not migrate. In elasmobranchs, unlike teleosts, Leydig cells are either absent from the interstitial tissue or rare and undifferentiated and their role in steroid production is at best marginal. While many studies have focused on topographical and functional interactions between the diverse cell types present in mammalian testis, only a few studies have brought particular attention to these aspects in fish. In fish, like in mammals, testicular cell-cell interactions are based on structural elements and chemical factors. Occasionally, various adhering junctions have been observed, essentially in teleosts, between Sertoli cells, between Sertoli cells and germ cells, between germ cells themselves, and interstitial cells. Furthermore, in some teleost species, using horseradish peroxidase or lanthanum salts, the presence of tight junctions between Sertoli cells has been correlated to the occurrence of a Sertoli barrier. In these species, the barrier develops after meiosis so that only haploid germ cells are shielded from the vascular system. In fish, recent development of techniques which enable the preparation and in vitro culture of enriched populations of testicular cells and of spermatocysts, has allowed investigations on functional aspects of cell-cell interactions. In particular, data have been obtained, in the trout, on the control of spermatogonia proliferation by Sertoli cell-conditioned media and, in the dogfish, on the steroidogenic activity of Sertoli cells, in relation to the differentiation stage of the associated germ cells. Furthermore information exists, in the trout, showing that intratubular macrophages may participate in the re-initiation of spermatogonial proliferation. In conclusion, the cytoarchitecture of fish testis, as compared to that of mammals, presents original features which provide unique opportunities to develop fruitful studies for a better understanding of the complex control mechanisms underlying testicular function in vertebrates.

The interstitial cells of the trout testis (Oncorhynchus mykiss): ultrastructural characterization and changes throughout the reproductive cycle.

The major non-vascular cell types present in the interstitial compartment in trout testes have been ultrastructurally characterized and cell changes in the course of the two first reproductive cycles have been studied. Three major cell types are always present fibroblasts, myoid cells and Leydig cells. Their structure varies with the maturational stage of the gonad. Fibroblasts are centrally located in the interstitial areas. Numerous typical myoid cells are always present near the basal lamina. However, at the end of a cycle some of them display degenerative changes, then disappear. At the beginning of the next cycle, fibroblasts appear to differentiate to new myoid cells. Leydig cells present before the start of the first spermatogenesis are replaced by new ones which probably also arise from fibroblasts. These cells progressively differentiate during spermatogenesis, so that most of the Leydig cells present during the spermiation phase are fully-differentiated steroidogenic cells. At the end of a cycle, a certain number of the Leydig cells disappear and are replaced at the beginning of the next cycle by new ones most likely derived from fibroblasts. The remaining Leydig cells dedifferentiate to cells which may redifferentiate during gonadal activity. Macrophages are present mostly at the end of a cycle and participate at this time in the removal of dead interstitial cells. In conclusion, at least some fibroblast-like cells are precursor cells, able to replace myoid cells and Leydig cells which disappear between two consecutive reproductive cycles.

Thermal acclimation and dietary lipids alter the composition, but not fluidity, of trout sperm plasma membrane.

The effect of a long-term adaptation of rainbow trout to 8 and 18 degrees C combined with a corn oil- or a fish oil-supplemented diet on the characteristics of the spermatozoan plasma membrane was investigated. The experiment lasted up to 22 mon during which spermatozoa were collected from the mature males. Spermatozoan plasma membranes were isolated by nitrogen cavitation, and the cholesterol content, phospholipid composition and fatty acid pattern were investigated. Membrane viscosity was assessed on whole cells by electron spin resonance using spin-labeled phospholipids. Neither diet nor rearing temperature influenced the cholesterol content of the plasma membrane nor the phospholipid class distribution. The rearing temperature of the broodstock only slightly affected the phospholipid fatty acids. A minor decrease in 18:0 and increase in monounsaturated fatty acids was observed for the cold-adapted fish. These modifications were not sufficient to affect membrane fluidity, and we conclude that trout spermatozoa do not display any homeoviscous adaptations in these conditions. On the contrary, the dietary fatty acid intake greatly modified the fatty acid profile of plasma membrane phospholipids. The fish oil-fed trout displayed a much higher n-3/n-6 fatty acid ratio than did the corn oil-fed ones, but the 22:6n-3 levels remained unchanged. Modifications in plasma membrane composition by the diet were obtained although neither of the two diets was deficient in essential fatty acids. The enrichment in n-3 fatty acids, however, did not affect plasma membrane fluidity which was unchanged by the diets.

Insulin-like growth factor-I and -II binding and action on DNA synthesis in rainbow trout spermatogonia and spermatocytes.

Radio-labeled recombinant human insulin-like growth factor (125I-rhIGF-I) bound specifically to total testicular cells and to spermatogonia plus primary spermatocytes (Go+CI) that had been prepared from trout testes at various maturation stages by centrifugal elutriation and then cleared of somatic cells by preculture in the presence of 2% Ultroser G. Binding sites showed high affinity (Ka = 0.5 +/- 0.2 x 10(10) M-1) and low capacity (1.1 +/- 0.8 fmol/10(6) testicular cells) for 125I-IGF-I. (Gln3, Ala4, Tyr15, Leu16)IGF-I ([QAYL]IGF-I) was equipotent to IGF-I in competing with 125I-IGF-I for site occupancy on Go+CI. Insulin-like growth factor-II (IGF-II) was to 10-fold less potent than IGF-I or (QAYL)IGF-I, while bovine insulin competed only about 300-fold higher concentrations. Go+CI were cultured for 3 days in the presence or absence of mammalian IGF-I, IGF-II, (QAYL)IGF-I, and salmon or bovine insulin. All these molecules stimulated the incorporation of [3H]thymidine (added during the last 24 h in culture) by Go+CI in a dose-dependent manner. The mean ED50, independent of testicular maturation stage, was 5.9 +/- 4.9 ng/ml and 29.1 +/- 15.8 ng/ml for IGF-I and IGF-II, respectively. (QAYL)IGF-I was as potent as IGF-I. Concentrations of salmon or bovine insulin 100- to 300-fold higher were required to produce effects similar to those of IGF-I. While recombinant human IGF binding protein (IGFBP-3) had no effect by itself of basal [3H]thymidine incorporation, it inhibited the effect of IGF-I in a dose-dependent manner; however, it had no effect on the stimulation by (QAYL)IGF-F. Although combinations of low concentrations of IGF-I and IGF-II or salmon insulin had additive effects, combinations of maximum concentrations did not. We conclude that, in vitro, IGFs stimulate DNA synthesis of trout male germ cells by interacting directly with these cells through one IGF receptor.