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[Effect of an analog of proline (L-azetidine-2-carboxylic acid) on in vitro differentiation of the rat fetal testis].

The initial stages of the development of the seminiferous cords involve the differentiation and the aggregation of primordial Sertoli cells opposite to cells which acquire a mesenchymal-like aspect. The hypothesis that the development of the seminiferous cords depends on epithelial-mesenchymal relations between the two cell types was submitted to experimental test. Male gonadal primordia of rat fetuses were cultured in vitro in a synthetic medium containing the proline competitor, L-Azetidine-2-Carboxylic Acid. This drug is known to disturb the synthesis and secretion of collagen and proline-containing proteins. It prevents testicular organogenesis or destroys it if it has begun. It suppresses the expression of laminin and fibronectin in the gonadal primordium. These observations are taken as evidence that cellular correlations of the epithelial-mesenchymal type play a role in the development of the testis as they do in that of other organs.

Animals

[Seminoma of testis. Analysis of failures and development of therapeutic strategies. Apropos of a Lyons series of 117 cases].

From Jan.61 to Dec.81, 117 patients with seminoma of testis were treated in the Leon Berard Centre, Lyon. All had undergone lymphography during investigation of possible extension, 19 were treated with 200 KV up 1966, 64 with Cobalt up to 1978 and 29 with photons x of 18 MV since that date. From 1979 adjuvant chemotherapy has always included cisplatinum. The 5 years survival rate was 95% of stage I (51/54 cases), 72% of stage II (26/36 cases) and 1/7 of stage III. Unsuccessful treatment of neoplasm was noted in 23 patients, in 80% of cases during the first two years and involving mainly pulmonary metastases. Three patients had mediastinal metastases while recovery surgery was possible in 4 cases. Three fatal iatrogenic complications were observed. Since the use of high energies, particularLy x beams of 18 MV there has been almost total absence of radic complications. Therapy now proposed is as follows: stage I: surgery plus radiotherapy; stage II A-B: surgery and irradiation avoiding mediastinum; stage II C and III: primary chemotherapy.

Adult

Gonadal dimorphism explained as a dosage effect of a locus on the sex chromosomes, the gonad-differentiation locus (GDL).

In human somatic cells bearing two X chromosomes, one X is genetically inactivated throughout most of its length, whereas in cells with one X and one Y both sex chromosomes are active (with the exception of the constitutive heterochromatin of the Y that is inert). The vast base of information concerning normal and abnormal human sexual development that has accumulated since the advent of human cytogenetics 3 decades ago can be integrated by the following hypothesis: Homologous gonad-differentiation loci (GDLs) exist on the X and Y. The GDLs are strictly sex-linked; that is, normally they do not recombine during spermatogenesis, so that considerable divergence in DNA sequence doubtless has occurred between the locus on the X and the locus on the Y. The abundance of their evolutionarily conserved product--a substance still to be identified--determines the path of differentiation that the indifferent gonadal anlage of the early embryo will take: if only one GDL is transcribed, the case when two X chromosomes are present, ovary will develop; if two GDLs are transcribed, the case when a Y is present along with an X, testis will develop. By implication, facultative X inactivation is an integral and essential component of the system adopted in mammalian evolution for accomplishing gonadal--viz., sexual--dimorphism.

Chromosome Mapping

[Arteriographic diagnosis of a tumor arising from an undescended testis].

A case with a tumor developed in an undescended testis diagnosed by arteriography is shown. Radiological diagnostic procedures in the diagnosis of cryptorchism are discussed. It is established that selective testicular arteriography is of great value in the preoperative diagnosis of an intra-abdominally located tumor developed in an undescended testis even in the area of modern high resolution non invasive imaging modalities.

Adult

Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme.

There are two isozymes of angiotensin-converting enzyme (ACE), one produced by somatic tissues and a smaller protein synthesized by developing spermatozoa (testis ACE). To investigate the molecular control of testis ACE, we generated mice transgenic for a construct containing a putative testis-specific ACE promoter linked to the Escherichia coli reporter gene encoding beta-galactosidase. The transgenic mice express beta-galactosidase protein and RNA only within the testis. Histochemical analysis of the transgenic mice shows co-localization of beta-galactosidase protein and endogenous ACE within elongating spermatozoa. These studies demonstrate that transcription of testis ACE is controlled by a strong intragenic testis-specific promoter that is contained within a 698-base pair fragment immediately upstream from the transcription start site of testis ACE. Characterization of the testis ACE promoter may provide insights into the molecular mechanisms controlling cell stage-specific gene expression in the male germ line.

Animals

Quantitation of testicular and somatic cytochromes c in testis and somatic tissues from developing rats.

By combining chromatographic and spectral procedures, simple and quantitative assays for somatic cytochrome c (cyt cs) and testicular cytochrome c (cyt ct) in crude animal tissue extracts were developed. Using this assay procedure, limited developmental studies of cyt ct and cs were performed with tissue extracts of 27-, 58-, and 85-day-old rats. Specific contents of cyt cs in somatic tissues (i.e., micrograms of cyt c/g of tissue) of these three age groups did not show significant variations. However, the amounts of both cyt ct and cs in testis were markedly increased as the rats approached maturity. Increasing cyt ct/cyt cs ratios as the rat developed to maturity suggest that expression of cyt ct is preferentially required for specific function of testis. Application of both molecular biological techniques and this assay (for holo-cyt ct) should be useful to study the overall regulation of the expression of cyt ct in testis.

Aging

Androgen binding protein as a biochemical marker of formation of the blood-testis barrier.

Androgen binding protein (ABP) was measured during postnatal development in normal and irradiated rats to determine whether development of a blood-testis barrier and formation of a continuous lumen from testis to epididymis is correlated with entry of ABP into the caput epididymis. ABP is found in normal testis as early as 14 days postnatally (0.2 pmol/mg), at which time no blood-testis barrier is observed by the peroxidase perfusion technique. Previous findings have shown a close correlation of blood-testis barrier development and lumen formation. Indeed, ABP is not detectable in the epididymis until 18-20 days of age (1.0 pmol/mg) at which time blood-testis barrier formation and lumen development is complete. Whole body irradiation (125 rads) of pregnant rats at 19-20 days of gestation produces male offspring with seminiferous tubules remarkably free of germinal epithelium and containing essentially only Sertoli cells. These Sertoli cell-enriched (SCE) testes produce normal amounts of ABP between 14 and 21 days postnatally. However, between 21 and 30 days of age the specific activity of ABP is significantly higher in the SCE tests (2.8 pmol/mg protein) than in normal testis (0.5 pmol/mg protein). In the SCE testis neither blood-testis barrier development nor lumen formation are complete until 30 days of age, at which time ABP is first detectable in epididymis of the irradiated rat. Thereafter there is a gradual decline of ABP in the SCE testis-and a dramatic increase in the epididymis. NIH-FSH-S-10 (200 mug/rat) injected SC into 14-day-old normal rats stimulated ABP in the testis from control levels of 0.15 pmol/mg to 1.46 pmol/mg within 4 h after injection. However, no ABP was detectable in the epididymis of either the control or the FSH-stimulated rats. These findings suggest that entry of ABP into caput epididymis is an index of blood-testis barrier formation and lumen development.

Androgens

Isolation and partial characterization of basic fibroblast growth factor from bovine testis.

A basic fibroblast growth factor (FGF) has been purified to homogeneity from bovine testis, using ammonium sulfate precipitation of the crude extract followed by three chromatographic steps, involving cation-exchange, heparin-Sepharose, and reversed-phase HPLC. Gas-phase sequence analysis showed the amino-terminal amino acid sequence of the isolated polypeptide as His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-, which is identical to the amino-terminal of the (16-146) fragment of basic FGF previously characterized from corpus luteum, adrenal, and kidney. The purified FGF was shown to have the same biological activity as that of basic FGF (1-146). This finding suggests that basic FGF is present in testis and may act as a local regulator of testicular function. In addition, testicular FGF might play an important role in spermatogenesis and/or the development of testis.

Amino Acid Sequence

Age-related effects of transection of the testicular blood vessels on subsequent testicular development in the pig.

Pigs of different ages were studied to assess the effect of age on the development of the testes after intra-abdominal transection of the testicular artery and vein. Transection of the testicular artery and vein at different stages of sexual development had a variable effect on the growth and differentiation of the testes. Transection performed at a young age (2 months) had no effect on the development of the testes (previous study). The animals used in this study were between 3 and 12 months (adult) at the time of surgical intervention. Their testes appeared to be more sensitive to disturbance of the main blood supply. The alternative vascular pedicle (vasal artery) was unable to adapt sufficiently to the increased physiological requirements of the testis during development in more than 50% of the animals. The results obtained from adult males indicate that intra-abdominal transection of the testicular vessels invariably has a deleterious effect on the testis.

Age Factors

Both L-Lactyl and D-Lactyl Enantiomers Modify Histones in Mouse Testis.

Dynamic histone posttranslational modifications are crucial to precisely orchestrate gene expression programs. The recently discovered histone lysine lactylation has already been explored in various pathological contexts, but less in normal tissues. This modification exists as two enantiomers, L- and D-lactylation; the former may more likely modify histones due to abundant L-lactate produced by glycolysis. Here, we report the identification by proteomics of L- and D-lactylation on lysines of histones H3 and H4 in mouse testis. We developed a targeted proteomic analysis of histone peptides using synthetic sequences modified by L- or D-lactyl, to acquire reliable identification and quantification data. Some histone peptides bearing either enantiomer are separated by reversed-phase chromatography. Interestingly, despite the fact that L-lactate is much more abundant than D-lactate in mouse testis, we estimated abundance ratios of L-over D-lactylation to lie between 0.4 and 1.6 on seven residues of histones H3 and H4. Next, targeted proteomic analyses were performed on histones extracted from meiotic and postmeiotic male germ cells (spermatocytes and round spermatids, respectively), which are known to use L-lactate as a main source of energy. Nonetheless, residues 18 and 23 of histone H3 (H3K18 and H3K23) were reliably quantified and shown to harbor balanced amounts of both enantiomers. The stoichiometry of lactylation is low over the whole sequence of H3 and H4, representing about 0.01 to 0.44%: this contrasts with acetylation which exists at up to 25 to 35% relative abundances on some N-terminal lysines. Yet, lactylation appears to be more abundant than acetylation on the C-terminal half of H3 and H4, where the latter modification is scarce. Collectively, our results suggest a mechanism producing a mixture of the two enantiomers of lactate, or of a more direct substrate for lactylation, that leads to the modification of histones by L- and D-lactylation.

Animals

Subunit structure of casein kinase II from bovine testis. Demonstration that the alpha and alpha' subunits are distinct polypeptides.

The relationship between the alpha and alpha' subunits of casein kinase II was studied. For this study, a rapid scheme for the purification of the enzyme from bovine testis was developed. Using a combination of chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite, gel filtration on Sephacryl S-300 and heparin-agarose, the enzyme was purified approximately 7,000-fold. The purification scheme was completed within 48 h and resulted in the purification of milligram quantities of casein kinase II from 1 kg of fresh bovine testis. The purified enzyme had high specific activity (3,000-5,000 nmol of phosphate transferred per min/mg protein) when assayed at 30 degrees C with ATP and the synthetic peptide RRRDDDSDDD as substrates. The isolated enzyme was a phosphoprotein with an alkali-labile phosphate content exceeding 2 mol/mol protein. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis three polypeptides were apparent: alpha (Mr 45,000), alpha' (Mr 40,000), and beta (Mr 26,000). Several lines of evidence conclusively demonstrated that the alpha and alpha' subunits are distinct polypeptides. Two-dimensional maps of 125I-tryptic peptides derived from the two proteins were related, but distinct. An antipeptide antibody was raised in rabbits which reacted only with the alpha subunit on immunoblots and failed to react with either the alpha' or beta subunits. Direct comparison of peptide sequences obtained from the alpha and alpha' subunits revealed differences between the two polypeptides. The results of this study clearly demonstrate that the alpha and alpha' subunits of casein kinase II are not related by post-translational modification and are probably encoded by different genes.

Amino Acid Sequence

Cancer of the undescended or maldescended testis.

An analysis of 45 cryptorchids (by history or examination) with a testicular cancer treated at Memorial Hospital, between 1934 and 1973, is presented. Twenty-five patients had the cryptorchid state repaired at ages four to 27 years, either spontaneously or by orchiopexy or hormonal therapy. Ipsilateral (24) or contralateral (one) intrascrotal testis tumors developed four to 47 years later. Twenty cryptorchid patients presented with ipsilateral inguinal (eleven), abdominal (seven), or contralateral intrascrotal (two) tumors. There were 18 pure seminomas, 17 embryonal carcinomas, nine teratocarcinomas, and one reticulum cell sarcoma. Five year survival rates as estimated by the product-limit method were 60% for the unrepaired cases and 41% for the repaired cases. The survival seems to follow histologic type and anatomical stage, whether the testis is within the scrotum or not. Five year survival similarly estimated was 78% in the seminomas and 29% in the other tumors. Twelve of thirteen survivors (including nine with seminoma) received postoperative irradiation to the regional lymphatics and eleven were without recurrent tumor for periods ranging from six to 28 years.

Adolescent

Newborn and immature rat testes contain gonadotropin-releasing hormone (GnRH) receptors, and their testosterone production is stimulated by a GnRH agonist in vitro.

The possibility of direct gonadal effects of GnRH in the newborn and immature rat testis was studied by two approaches: the presence of specific high-affinity receptors for a GnRH agonist analogue (Buserelin) was studied in animals between the ages of 1 and 60 days; and the effect of Buserelin on testicular testosterone production was studied in vitro. GnRH receptors were found in the testis tissue at all ages studied, and there was a gradual increase in the number of binding sites per testis with advancing age. The highest concentration of GnRH receptors was found in the rat testis tissue on day 1 of life, 3.2 +/- 0.4 fmoles/g tissue (mean +/- SE, n = 5). The binding declined to a nadir, 1.5 +/- 0.5 fmoles/g tissue (in = 10), on day 15 post-partum (p less than 0.05), and thereafter increased again gradually to a level of 2.7 +/- 0.3 fmoles/g (n = 5) on day 60 of life (p less than 0.05). The biphasic concentration curve of GnRH receptors followed closely the volume density of Leydig cells in the developing rat testis. Buserelin at a concentration of 10(-7) M was able to stimulate significantly (p less than 0.01), by 98-146%, the testosterone production of decapsulated testes at all ages studied between 1 and 60 days of life. These results indicate the presence of functional GnRH receptors in the testis tissue of newborn and immature rats.

Animals

Immunocytochemical localization of bioregulatory peptides in marmoset testes.

Immunocytochemical localization of neuropeptides (beta-endorphin, substance P, arginine vasopressin, oxytocin), pituitary hormones (adrenocorticotropin, prolactin, growth hormone, follicle stimulating hormone (FSH), gonadal inhibin, gastrin, and human chorionic gonadotrophin (hCG)) was carried out in marmoset testis during development. Both intensity of immunostaining and distribution of these peptides in testicular compartments viz. seminiferous tubules and Leydig cells changed dramatically during development. In vitro biosynthesis of inhibin and FSH was increased by hCG, whereas prolactin (5 micrograms) and prostatic inhibin peptide suppressed the synthesis of these hormones.

Aging

Structure of the right testis of sexually mature genetically female fowl experimentally masculinized during embryonic life and submitted to a posthatching left castration.

Posthatching left castration of genetically female fowl, Gallus domesticus, preceded, during embryonic life, by a masculinizing treatment associating a testis graft and an antiestrogen resulted in the development of the right rudimentary gonad into a testis. Examined after the sexual maturity, the right testis of most treated animals was entirely composed of seminiferous tubules possessing a spermatogenic cell complement. Spermiogenesis proceeded to the stage of spermatozoon in 4 out of 17 treated animals and was almost as well organized as in a normal cock testis in 3 of them. Testis development appeared then as clearly improved, compared to that described previously in only left-castrated, with or without treatment with an antiestrogen, or only sex-reversed female fowl. The possible mechanism of this improvement is discussed.

Animals