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Cytoplasmic filaments in fetal and neonatal pig testis.

Leydig cells in developing fetal pig testis contained during the fetal regressive phase large accumulations of intermediate filaments. Before and after this period these filaments were arranged in a criss-cross fashion. In the pig as well as in the dog testis these filaments have been characterized as vimentin. Within the vimentin aggregates occasionally a weak positive actin reaction was seen in pig but not in dog Leydig cells. Microfilaments were hardly observed. Most Sertoli cells contained a layer of actin microfilaments close to the basal cell membrane. In the lower cell compartment and around the nucleus (intermediate) vimentin filaments could be observed in a criss-cross configuration.

Aging

Development of the inhibitory guanine nucleotide-binding regulatory protein in the rat testis.

The functional development of the inhibitory guanine nucleotide-binding regulatory protein (Gi) and anti-diuretic hormone (ADH) activity was investigated in rat testes. Adult (90-day-old), adolescent (40-day-old), prepubertal (23-day-old), and fetal (20.5 days of gestation) testis cells were cultured with 100 ng/ml pertussis toxin for 24 h. The cells were then cultured with human chorionic gonadotropin (hCG), the ADH agonist arginine vasotocin (AVT), or a combination of the two. Testis cells from rats 23, 40, and 90 days of age that were incubated with hCG increased testosterone production when compared with controls. Preincubation of the cells from postnatal rats with pertussis toxin significantly increased hCG-stimulated testosterone secretion when compared to cells preincubated in medium only at all three ages. AVT suppressed hCG-stimulated testosterone secretion, but this suppression was partially reversed in cells from all postnatal ages preincubated with pertussis toxin. Fetal testis cells showed no response to preincubation with pertussis toxin, even when levels were increased to 400 ng/ml or when pertussis toxin treatment was continued throughout the culture period. AVT also had no effect on fetal testis cells. These results indicate that the Gi protein and AVT are not functional in fetal testes but are active from prepubertal stages of development through maturity.

Animals

Postnatal development of the tubular lamina propria and the intertubular tissue in the bovine testis.

The postnatal development of intertubular cells and vessels and of the tubular lamina propria was studied in three locations of perfusion-fixed bovine testes from 31 animals ranging from 4 to 78 weeks. The postnatal morphological differentiation of the testis is not uniform, regional differences have to be considered. The intertubular cell population is composed of mesenchyme-like cells, fibrocytes, Leydig cells, peritubular cells and mononuclear cells. In 4- and 8-week-old testes mesenchyme-like cells are the dominating element. These pluripotent cells proliferate by frequent mitoses and are the precursors of Leydig cells, contractile peritubular cells and fibrocytes. Morphologically differentiated Leydig cells are encountered throughout the entire period of postnatal development. In 4-week-old testes degenerating fetal and newly formed postnatal Leydig cells are seen in juxtaposition to each other. From the 8th week on, only postnatal Leydig cells are present. Between 16 and 30 weeks large-scale degeneration of prepuberal Leydig cells is observed. The Leydig cells that survive this degenerative phase constitute the long-lasting adult population. 20-30% (numerically) of all intertubular cells at all ages are free mononuclear cells. These are found as lymphocytes, plasma cells, monocytes, macrophages and light intercalated cells (LIC). The latter are monocyte-derived, Leydig cell-associated typical cells of the bovine testis. The differentiation of the two main components of the tubular lamina propria, (i) basal lamina and (ii) peritubular cell sheath, seems to be effected rather independent from each other and also from hormonal signals important for the development of the germinal cells. The laminated basal lamina reaches nearly 3 micron at 16 weeks and is later on continuously reduced. At 25 weeks the peritubular cells have transformed into contractile myofibroblasts. At this period the germinal epithelium is still in a prepuberal state.

Aging

Quantitative evaluation of the maintenance and development of spermatocytes and round spermatids in cultured tubule fragments from immature rat testis.

Maintenance and development of spermatocytes and round spermatids was studied in an in-vitro incubation system. This system consisted of open tubule fragments from 26-day-old rat testes, obtained after collagenase treatment. The tubule fragments contained Sertoli cells and spermatogenic cells up to and including a small number of early round spermatids. The number of primary spermatocytes and round spermatids in the tubule fragments was estimated using flow-cytometric analysis, immediately after isolation and after 72 h of incubation. In addition, the activity of LDH-C4 in the tubule fragments was measured. After 72 h of incubation, the percentage of spermatocytes was reduced by 70-80%, but the percentage of spermatids was doubled. The total LDH-C4 activity per well was increased 2-3-fold during 72 h of incubation of the fragments. A modest improvement of the culture results was observed when a combination of FSH, insulin, retinol and testosterone was added to the medium. LDH-C4 activity was investigated to see whether it could be used as a quantitative marker of isolated and cultured spermatocytes and spermatids. It was observed that LDH-C4 activity per cell was decreased when spermatocytes and spermatids were isolated and/or incubated at 4 degrees C. However, the cellular enzyme activity returned to control values during subsequent incubation of the cells at 32 degrees C, either in the absence or presence of a protein synthesis inhibitor. Cellular LDH-C4 activity may be influenced not only by temperature, but possibly also by other cell isolation conditions. It is concluded that LDH-C4 activity may not be a reliable quantitative marker for the presence of spermatocytes and spermatids in culture, but should be used in combination with other analytical methods such as DNA estimation and DNA flow cytometry.

Adenosine Triphosphate

Awareness of testicular cancer in New Zealand men.

Testicular cancer is the most common malignancy in young men and is frequently associated with delays in presentation. It has been postulated that ignorance and poor awareness of this type of cancer may contribute to such delays, which may result in high stage disease and subsequent treatment-related morbidity. This study was undertaken to assess the level of awareness of testicular cancer in a sample of 500 men from Wellington, New Zealand, and to specifically examine awareness related to the age, race, and socio-economic level of the respondents. Sixty-one per cent of men returned a satisfactorily completed questionnaire. Most (70%) were unaware that cancer of the testis could develop. Of those who correctly identified testis cancer as occurring, half recognized the most commonly affected age group, 40% understood testis cancer could be treated, and 14% believed testis cancer could be cured with treatment. The lowest awareness score (mean +/- s.d.) were found in the 18-30 age group. The score in this group (2.82 +/- 1.47) was significantly lower than that seen in the 31-40 (mean 3.66 +/- 1.81, P = 0.009) or 41-50 (mean 3.57 +/- 2.16, P = 0.036) age groups. Differences were also seen between ethnic groups, with Maori having significantly lower scores than non-Maori (Maori, mean 2.69 +/- 1.52; non-Maori, 3.45 +/- 1.83; P = 0.044). Higher scores were associated with higher socio-economic grouping. The overall poor awareness of men in this study supports the need for a public education programme aimed at encouraging men to seek early advice following the recognition of testicular masses.

Adolescent

A comparative study of the effects of non-union of testis and epididymis and cryptorchidism on testicular development and spermatogenesis in the rat.

Malunion of testis and epididymis is a congenital anomaly of human undescended testicles which may be of considerable clinical importance. In an experimental model in rat the effects of early disconnection of testis and epididymis (16 days) on testicular development have been testis and puberty. The results are compared with corresponding effects of cryptorchidism and sham-operation. The testes were weighed, and the relative proportions of haploid, diploid and tetraploid cells were quantified by DNA flow cytometry. The histology was evaluated by light and electron microscopy. The non-union operated testes showed a close to normal weight development until about 40 days of age, whereafter a decline occurred. The progression of the maturation division was only slightly reduced at 30 and 37 days of age, but later the spermatogenesis was significantly inhibited. At 58 days of age the maturation division had practically ceased in non-union operated testes. In cryptorchid animals the onset and progression of maturation divisions were almost totally depressed, and only traces of haploid cells were seen in some specimens. The transient increase in tetraploid cells, reflecting primary spermatocytes seen at about 30 days during normal testicular development, was neither depressed in non-union operated nor in cryptorchid animals. This indicates that the major block in spermatogenesis in both situations is at the stage of the primary spermatocytes. At 58 days of age the testicular histology was similar in non-union operated and cryptorchid testicles with many tubular sections showing only Sertoli cells. Scattered and partly degenerated germinal cells were seen in some sections. No histological signs of increased intratubular fluid pressure were detected, suggesting that pressure atrophy is not decisive for the reduced spermatogenesis in non-union operated gonads.

Animals

The contribution of the mesonephros to the development of the sheep fetal testis.

In this study, which was performed on 52 sheep fetuses aged 24 to 85 days, we examined the relationship between testicular development and the "giant" mesonephric nephron, a peculiar structure consisting of a large glomerulus and multiple tubules. The development of the testis occurred in two phases. The preparatory phase began at day 24 of gestation, evolved simultaneously with the involution of the glomerulus of the giant nephron, and was characterized by mobilization of the glomerular cells and by their colonization of the genital ridge. By day 31, a prominent mass of migrating mesonephric cells had developed; it extended uninterrupted from the giant glomerulus into the gonad where the mesonephric cells associated with the germinal cells forming the cellular template from which the testicular cords later became assembled. At complete involution of the glomerulus (day 52), the migratory mass implanted on the tubules of the giant nephron, which thus became continuous with the gonad. During the organizational phase, which began at day 29 with the formation of the tunica albuginea, the various components of this continuum became progressively organized along a testis-to-mesonephros direction into seminiferous cords, tubuli recti, the cords of the rete testis, and the ductules efferentes. These observations show that, in the sheep, the precursors of the Sertoli cells are mesonephric in origin, and that the genital tract proximal to the epididymis differentiates from a single mesonephric nephron.

Animals

Morphometric studies of development of the rat testis under physiological conditions and after CdCl2 poisoning.

The morphometrical data of the rat testis were studied between the 1st day and 1.5 year of life under physiological conditions and after intraperitoneal injection of a single dose of cadmium chloride (1.5 mg CdCl2/kg body weight). Under physiological conditions three main period of development occur: between the 1st and 30th day of life, between the 30th and 45th day of life, and after the 45th day of life. The greatest changes after the injection of cadmium were noticed in the period between the 15th and 30th day of life. The periphery of the organ was the first to suffer severe damage.

Animals

Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis.

BSAP has been identified previously as a transcription factor that is expressed at early, but not late, stages of B-cell differentiation. Biochemical purification and cDNA cloning has now revealed that BSAP belongs to the family of paired domain proteins. BSAP is encoded by the Pax-5 gene and has been highly conserved between human and mouse. An intact paired domain was shown to be both necessary and sufficient for DNA binding of BSAP. Binding studies with several BSAP recognition sequences demonstrated that the sequence specificity of BSAP differs from that of the distantly related paired domain protein Pax-1. During embryogenesis, the BSAP gene is transiently expressed in the mesencephalon and spinal cord with a spatial and temporal expression pattern that is distinct from that of other Pax genes in the developing central nervous system (CNS). Later, the expression of the BSAP gene shifts to the fetal liver where it correlates with the onset of B lymphopoiesis. BSAP expression persists in B lymphocytes and is also seen in the testis of the adult mouse. All of this evidence indicates that the transcription factor BSAP may not only play an important role in B-cell differentiation but also in neural development and spermatogenesis.

Amino Acid Sequence

Leydig cell development in the pig testis during the late fetal and early postnatal period: an electron microscopic study with attention to the influence of fetal decapitation.

The ultrastructure of fetal and postnatal pig Leydig cells was studied from 75 days postcoitum (p.c.) to 1 month after birth. Additionally, decapitated fetuses from 75 days p.c. until birth were used to study the effect of deprivation of gonadotrophins on the ultrastructure of Leydig cells. Normal Leydig cell development was characterized by a change in smooth endoplasmic reticulum (SER). Next to branched tubular SER, whirls of elaborate and tightly packed SER membranes appeared. The amount of SER increased with age but decreased slightly before the end of the observation period. Rough endoplasmic reticulum (RER) was a minor component. Large bundles or whirls of intermediate filaments were abundant until just before birth; thereafter, they decreased drastically. Peroxisome-like structures and crystalloid bodies were observed with increasing frequency from 75 days p.c. and 20 days postpartum onward. Polygonal lysosome-like dense bodies transformed into complex membranous structures especially after birth. Giant mitochondria occurred in the late fetal and postnatal period. From 75 days p.c. onward fully developed Leydig cells were scarce in testes of decapitated fetuses. Leydig cell characteristics disappeared toward the end of the fetal period; only the cell shape, the large bundles or whirls of intermediate filaments, some scarce polygonal lysosome-like dense bodies, and RER remained, but SER was negligible. Progressive hemorrhages apparent in situ were correlated positively with fetal age. The dependency of Leydig cells upon LH began between 60 and 75 days postcoitum.

Animals

Effect of embryonic and/or neonatal diethylstilbestrol and allylestrenol treatment on postnatal development of the chick testis.

The synthetic steroid diethylstilbestrol (DES) and the steroid-like allylestrenol (AE) have been used for years in human medicine for the protection of pregnancy. The hazards to the fetus of gestational DES treatment are well established [2, 17, 18]. Knowledge of a similar effect of AE is still fragmentary. Therefore, further studies are required of the after-effects of embryonic and perinatal AE exposure. In our earlier experiments with polypeptide hormones [4, 5, 6] we have observed that perinatal age is a critical period in the maturation of hormone receptors. In this period the presence of hormone induces the development of its specific receptors. The phenomenon is termed hormonal imprinting [4, 5, 6]. During its maturation the receptor is flexible and the presence of non-specific hormones capable of binding to it may alter its normal development Accordingly, even a single hormone injection in the perinatal period may alter the hormone-sensitivity of the target organ.

Allylestrenol

Thyroid-testis interrelationship during the development and sexual maturity of the rat.

The effects of thyroidectomy and treatment with thyroxine (T4) were studied in immature male rats to evaluate the role of the thyroid in the development of testicular functions. Thyroidectomy inhibited gametogenesis and development of the Leydig cell in these rats. However, the effects could be reversed by administration of 10 micrograms T4 (i.p.) daily for 30 days.

Animals

Insulin-like growth factor I in the developing and mature rat testis: immunohistochemical aspects.

The distribution of insulin-like growth factor I (IGF-I; somatomedin C) was mapped in testes of different aged rats by using immunohistochemical techniques. The antiserum used, K 624, has been demonstrated to be specific for human IGF-I, as defined by several criteria. Antibodies to the M1 subunit of ribonucleotide reductase, a key enzyme in DNA synthesis, were used to visualize meiotic and mitotic cells. Cytoplasmic IGF-I-like immunoreactivity as demonstrable during the first two postnatal weeks in spermatogenic cells, in Sertoli cells, and in Leydig cells. The IGF-I-like immunoreactivity decreased in the Sertoli and Leydig cells during the third and fourth postnatal weeks, and in adult rats, only spermatogenic cells showed IGF-I-like immunoreactivity. In mature rat testes, the spermatocytes were strongly immunoreactive. During puberty and adulthood, the spermatogonia expressed subunit M1 ribonucleotide reductase immunoreactivity, whereas no IGF-I-like immunoreactivity could be detected. No extracellular immunoreactivity was observed. We propose that IGF-I and/or IGF-I-like substances, possibly formed by primary spermatocytes, are likely to be involved in differentiation processes, but not in the initiation of cell proliferation in adult testes. The autocrine and/or paracrine action of IGF-I and/or IGF-I-like substances may thus have different action in developing testes than in adult testes. Our results do, however, not allow firm statements about whether IGF-I and related substances exert their actions on Sertoli cells or spermatogenic cells.

Aging

Acid phosphatases in the mouse testis: activity changes during development.

The histology and acid phosphatase activities of the developing testes in the mouse, from 4 days of age until maturity, were analyzed. The specific enzyme activity with p-nitrophenyl phosphate as substrate increased after 3 weeks of age and then reached a plateau. Cobalt and zinc markedly increased the specific activity after the third week. After fractionation the testicular homogenate revealed four acid phosphatases. Enzyme I maintained a high activity during the first 3 weeks but steadily declined thereafter. Enzyme II, present in all age groups, showed a moderate increase after the fourth week. Enzymes III and IV were low and declined further during the first 3 weeks with a subsequent increase. This occurred concomitantly with the appearance of spermatids and mature sperm cells. Changes in enzyme activities seem to reflect the alterations in cellular composition of the testis during the developmental process. Enzymes III and IV were probably associated with spermatids and sperm cells.

Acid Phosphatase

Expression profile of Ldh-a in the developing rat (Rattus norvegicus) testis suggests regulation at the translational level.

Expression of Ldh-a and Ldh-c mRNAs was examined in the rat testis. The mRNA levels of both Ldh-a and Ldh-c increase during testicular maturation. In the adult testis, Ldh-a mRNA is expressed maximally in primary spermatocytes. Comparison of the Ldh-a mRNA expression profile with its translation product suggests that this gene is translationally down-regulated during spermatogenesis.

Animals