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PubMed1991

[Nuclear triiodothyronine receptors in the testis of the growing rat are regulated by the thyroid status].

The effects of thyroid status on the binding capacity, association constant (Ka) and receptor occupancy during postnatal rat testis development were evaluated. Hypothyroidism (induced by oral administration of 0.05% methimazole from the day of birth) increased the total T3 binding capacity in the testis, retarding the normal developmental decrease in T3 receptor number (mean maximal binding capacities estimated by Scatchard analysis for 21-day-old eu- and hypothyroid rats were 117 and 173 fmol/mg DNA, respectively). The rat thyroid status also affected the percentage of T3 receptor occupancy but not the affinity of binding (as measured by Ka). The postnatal developmental changes in T3 binding capacity induced by hypothyroidism were completely reversed by T3 replacement. These results suggest that T3 nuclear receptors in the developing rat testis are modulated by thyroid hormones.

Animals↗

PubMed1991

Sex determination in mammals. How many genes are involved?

The genetic mechanisms of sex determination in mammals have not yet been clarified. Y-linked and X-linked zinc finger genes from humans and mice were recently cloned and characterized. The Y-linked zinc finger gene was originally thought to be the testis-determining factor. However, at the present time, it seems clear that this gene is not the master gene triggering the cascade of events leading to sex determination. The human testis-determining factor gene is known to be located in the nonhomologous region of the short arm of the Y chromosome, close to an Alu repeat that marks the boundary of the pseudoautosomal region. Lately, this region has been found to contain a new gene that is a strong candidate for the male-determining factor. Data on humans and on laboratory and vole mice showing abnormalities of the sex determination mechanisms indicate that testis development depends on the presence of a testis-determining factor gene functioning in cooperation with X-linked and autosomal genes. Ovary development would depend on the absence of the testis-determining factor and perhaps on an alternative splicing of the transcripts from autosomal and X-linked genes involved in sex determination.

Animals↗

PubMed1995

Apoptosis in testis germ cells: developmental changes in gonadotropin dependence and localization to selective tubule stages.

Recent studies have demonstrated apoptotic DNA fragmentation in the testis of immature rats deprived of gonadotropins. However, the exact cell type undergoing apoptosis during testis development and the age differences of gonadotropin dependence of testis cell apoptosis are unclear. The present study used gel fractionation and in situ methods to quantitate developmental changes of testis cell DNA fragmentation and to localize the specific cell type affected in developing rats with and without treatment with a GnRH antagonist. Apoptotic DNA fragmentation in whole testis was measured in rats between 8-70 days of age. A gradual increase (1.8- to 2.0-fold) in testis apoptotic DNA fragmentation was seen in rats between 16-28 days of age, compared with 8-day-old animals, followed by a decrease in adult animals. To study gonadotropin dependence of testicular apoptosis, serum FSH and, to a lesser extent, LH were suppressed by treatment with a long-acting GnRH antagonist (azaline-B, 250 micrograms/kg body wt, two injections at 2-day intervals). Pretreatment with the GnRH antagonist increased apoptotic DNA fragmentation in rats between 16-32 days of age but not in younger and adult animals demonstrating an age-related change in gonadotropin dependence. To identify the exact testis cell type undergoing apoptosis, in situ analysis of DNA fragmentation was performed. In rats at 16-24 days of age, spermatocytes in selected tubules were found to have increased DNA fragmentation. In contrast, neither Leydig cells nor Sertoli cells were affected. In 32-day-old and adult animals, increased DNA fragmentation was seen in early primary spermatocytes of some tubules. Treatment with GnRH antagonist increased the number of cells with DNA fragmentation as well as percentage of tubules affected. In animals between 16-32 days of age, meiotic spermatocytes were labeled, whereas early spermatids were also labeled in 24- and 32-day-old animals. In adult animals, the level of apoptotic DNA fragmentation was not affected by GnRH antagonist treatment. However, DNA isolated from specific stages of the seminiferous tubules of adult animals showed stage-specific changes of apoptotic DNA fragmentation with 2-fold higher levels found in stages I and XII-XIV compared with stage VIII. In situ analysis of adult testis demonstrated that spermatocytes were the major cell type affected. In conclusion, the present study demonstrated that at least three factors determine the onset of apoptosis of the male germ cells: 1) the developmental stage of the animal; 2) serum levels of gonadotropins, especially FSH; and 3) specific stage of the seminiferous epithelial cycle. The present approach provides the basis for future analysis of the role of gonadotropins and other factors in the regulation of testis cell degeneration in normal and pathological states.

Animals↗

PubMed1990

[The influence of chemotherapy on testicular function of boys with acute lymphoblastic leukemia].

In order to investigate gonadal development of boys after chemotherapy, testicular biopsy specimens, which were obtained from 16 boys within 6 months after completion of the therapy for acute lymphoblastic leukemia, were assessed and their gonadal function was examined. More than half of them showed a decrease of Mean Tubular Diameter and Johnsen's Score Count, but no specimen showed a decrease of Tubular Fertility Index. Seventeen healed patients consisting of 12 biopsied and 5 non-biopsied cases were examined as to the volume of testis, development of the genital organ, skeletal age, plasma LH, FSH, testosterone, LH-RH test and HCG test. The period of follow-up after completion of chemotherapy varied from 1 month to 5 years. All patients showed testis volume, development of external genitalia and skeletal age suitable for their age. Some patients who were examined within 2 years after completion of chemotherapy, showed abnormal endocrine functions, but other patients examined after more than two years showed normal endocrine functions except a case who received testicular irradiation. Semen analysis in one case revealed density and motility within normal range. These data indicate that chemotherapy of acute lymphoblastic leukemia in boys damages testicular function, but more than 2 years later the spermatogenesis as well as endocrine function is expected to recover gradually.

Adolescent↗

PubMed1994

Photoperiod regulates testis cell apoptosis in Djungarian hamsters.

Reproductive activity in the Djungarian hamster is controlled by seasonal variations in day length. Exposure to long days stimulates testis development, while exposure to short days induces testis regression. We recently found that testis regression after gonadotropin deprivation in rats is associated with increases in apoptosis. Here we sought to determine whether or not apoptosis is associated with the testis regression and/or recrudescence that occurs naturally in seasonally breeding mammals. Newborn male hamsters were maintained on long days (16L:8D) until 3 wk of age before being transferred to short days (8L:16D). Following decreases in serum FSH within 3 days of exposure to short days, testis weight decreased by 52% at Day 10, reaching a 70% decrease after 21 days. Analysis of testis cell DNA fragmentation showed a 4.9-fold increase of low-molecular-weight DNA as early as 5 days after transfer to short days; this was followed by a time-dependent decrease. The observed increases in testis cell apoptosis were correlated with decreases in serum testosterone, but decreases in Leydig cell LH receptor content were delayed. In a second study, 6-wk-old hamsters with regressed testes due to a 3-wk exposure to short days were transferred back to long days. After increases in serum FSH within 3 days of photostimulation, a 2-fold elevation in testis weight was found at Day 5. The increase in testis weight was associated with a 65% decrease of testis apoptosis within 5 days of photostimulation. Also, increases in serum testosterone and LH receptor content were observed after 5 and 10 days of exposure to long days, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1997

beta-Hexosaminidase immunolocalization and alpha- and beta-subunit gene expression in the rat testis and epididymis.

beta-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G(M2) gangliosidoses. beta-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for beta-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the alpha- and beta-subunits of beta-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. beta-hexosaminidase alpha-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, beta-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7-91 postnatal days of age, testis levels of alpha-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, beta-subunit mRNA was expressed at low levels throughout tests development. In isolated male germ cells, beta-hexosaminidase alpha-subunit expression was most abundant in haploid round spermatids, whereas the beta-subunit mRNA was not detected in germ cells. Within the epididymis both alpha- and beta-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that beta-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, beta-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that beta-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of beta-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions.

Animals↗

PubMed1997

An mRNA differential display strategy for cloning genes expressed during mouse gonad development.

The mRNA differential display technique has become a popular method for isolating novel genes in a variety of biological systems including carcinogenesis, hormone regulation, plant biology and neurobiology. We have further developed the method by optimizing different steps for the use of small amounts of material, such that differential display can be used in the study of developmental biology. Our techniques include a new assay for elimination of false positive cDNA clones and a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method for the rapid analysis of differences in gene expression. This improved mRNA differential display strategy requires less than 4 microg of total RNA. We have used it for the isolation of genes which are expressed during gonad development in the mouse. One of the cDNAs found, cDNA 4.3 which corresponds to a part of the gene encoding the steroid hydroxylase 3betaHSD I, was shown to be a valuable marker for adrenal development and for Leydig cell differentiation and organization during testis development.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Changes in the peritoneum during the development of the testis, epididymis and ductus deferens in the pig.

The development of the peritoneal folds of the testis, epididymis and ductus deferens of the pig prior to testicular descent was studied in 18 to 82 days-old embryos/fetuses. The parietal attachment of the mesonephros, mesonephric and paramesonephric ducts and gubernaculum constituted the urogenital mesentery. This could be divided in a cranial and a caudal part. The first fixed the mesonephros and had a mesogonad as a secondary fold. The second had two branches, one laterally to the umbilical artery belonging to the gubernaculum (Plica gubernacularis) and the medial to the artery contained the meso- and paramesonephric ducts. The mesogonad was related caudally with Plica gubernacularis. The fold of the meso- and paramesonephric ducts could also be divided in two parts. The caudal was a primary component of the urogenital mesentery, while the cranial was first ventral and later lateral to the mesogonad (mesorchium) and appeared as a secondary fold of the urogenital mesentery (cranial part). A vascular fold contributed to separate the suspensory ligament of the testis from the developing mesorchium. The proposed meaning of the terms used might be useful for comparative studies.

Animals↗

PubMed1990

The testis and tissue transplantation: historical aspects.

Transplantation experiments involving the testis have been performed since the days of John Hunter, who transplanted a testis into the belly of a hen. The first person to use the testis as a site of transplantation appears to have been Sand, who found in 1919 that an ovary transplanted into the substance of the testis developed follicles. By 1970, there was considerable evidence that the testis under some circumstances was a relatively favorable site for graft survival. However, much of the evidence was equivocal, and the immunological privilege was by no means complete.

Animals↗

PubMed1981

Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse.

Total mouse testis RNA directs the synthesis of the sperm-specific C subunit of lactate dehydrogenase-X (LDH-X) when translated in a cell-free system derived from rabbit reticulocytes. The newly synthesized C subunits were isolated by immunoprecipitation with antibody specific for this isozyme, and quantitated by electrophoresis on SDS polyacrylamide gels. The amount of radioactivity incorporated into the enzyme subunit was directly proportional to the amount of testis RNA added to the translational system, thereby providing a sensitive and reliable method for assessing relative LDH-X mRNA activity. A combination of sucrose gradient centrifugation and oligo(dT)-cellulose chromatography resulted in a 23-fold purification of LDH-X mRNA over total cytoplasmic testis RNA. Analysis of LDH-X mRNA activity in the developing testis indicated that the appearance of functional LDH-X mRNA activity coincides with the appearance of LDH-X catalytic activity at 14 d postpartum. Measurement of LDH-X mRNA levels in separated testis cell populations prepared by centrifugal elutriation demonstrated that LDH-X mRNA represents 0.17-0.18% of the total functional mRNA activity in fractions enriched in pachytene spermatocytes and round spermatids, but only 0.09-0.10% of the translation products of elongated spermatids.

Animals↗

PubMed1991

Quantitative analysis of the development of genital organs from the urogenital sinus of the fetal male mouse treated prenatally with a 5 alpha-reductase inhibitor.

The role of 5 alpha-dihydrotestosterone (DHT) in the development of the genital organs and in the differentiation of the genital tract into prostate, coagulating gland (CG), bulbo-urethral gland (BUG) and seminal vesicle (SV) in male mice exposed prenatally to the 5 alpha-reductase inhibitor 6-methylene-4-pregnene-3,20-dione (6-MP) has been examined quantitatively. Female ICR mice were given 7 daily s.c. injections of the inhibitor (400 mg/day) starting on day 12 of gestation and the experiment was terminated on day 19 when the fetuses were removed by Caesarean section. In the prenatally 6-MP-exposed male mice the anogenital distance was significantly shorter than in the controls. Feminization of the nipples and hypospadias of the phallic urethra were noted. Development of prostate, CG and BUG was significantly suppressed. SV and testis development were not affected. These results lend further support to the conclusion that DHT is necessary for the development of the urogenital sinus (prostate, CG and BUG) and penis, and for the regression of the nipples in male mice. Reproductive abnormalities were not found in 90-day-old mice of both sexes exposed to 6-MP in utero. The 6-MP-exposed male and female mice had a normal reproductive capacity when mated with normal mice. These results show that 6-MP-induced growth retardation of reproductive organs is evident on day 19 of gestation, but that such retardation is no longer apparent in the adult.

5-alpha Reductase Inhibitors↗

PubMed1994

Immunohistochemical demonstration of cytoskeletal proteins in the ovine testis during postnatal development.

The distribution pattern of actin, desmin, vimentin and tubulin in the ovine testis during postnatal development was investigated by means of immunohistochemical methods. The postnatal development of the ovine testis can be divided into five phases. Phases I through III represent the prepubertal period, phase IV puberty and phase V the postpubertal adult stage. In peritubular cells alpha-smooth muscle actin is present, its amount increasing with advancing age of the animals. Structural F-actin is localized in peritubular myoid cells and Sertoli cells, of the adult testis. In Sertoli cells structural F-actin-positive material is observed at the level of the Sertoli-Sertoli junctions, at contact sites of Sertoli cells with primary spermatocytes and in the immediate vicinity of elongating spermatid heads during the acrosome phase of spermiogenesis. Desmin is present in intertubular and peritubular cells during the early prepubertal period, but vanishes completely as soon as the animals reach puberty. Vimentin is present in the cytoplasm of prespermatogonia I, but disappears when these change into prespermatogonia II. In prepubertal supporting cells the vimentin content increases, and in the adult the positive filament bundles create a flame-like pattern around the unstained nucleus. Cyclical variations during the seminiferous epithelial cycle are not observed. Expression of alpha-tubulin is found in the cytoplasm of prespermatogonia I and to a lesser extent in prespermatogonia II and spermatogonia. The immunoreaction is also seen in the microtubules of the axonema and manchette of elongating spermatids. The histochemical demonstration of the high alpha-tubulin concentration in supporting and Sertoli cells is an excellent method for studying changes of cellular shape and size during ontogenesis as well as during the seminiferous epithelial cycle.

Actins↗

PubMed1996

Molecular determinants of sexual differentiation.

The processes of sexual differentiation have been greatly clarified by molecular biologic discoveries over the past five years. Gonadal differentiation into a testis or ovaries is controlled by a multitude of genes beginning with SRY which is believed to represent the testis determining factor. Other genes involved include SF-1, WT-1, DAX-1, and SOX9. The fully developed testis produces Mullerian inhibiting substance and testosterone to create the male phenotype; the female phenotype develops in their absence. This hormonally-driven process also requires additional factors and appropriate receptors. Errors in this pathway may be manifested clinically as intersex disorders, and the study of these disorders has helped to further elucidate the molecular mechanisms of sexual differentiation.

Animals↗

PubMed1993

Follicle-stimulating hormone induction of steel factor (SLF) mRNA in mouse Sertoli cells and stimulation of DNA synthesis in spermatogonia by soluble SLF.

Follicle-stimulating hormone (FSH) and its intracellular mediator, cAMP, increase the mRNA levels for the Steel factor (SLF, the c-kit ligand) in cultured primary mouse Sertoli cells. The inductive effect of cAMP is more evident in cultures from 13-day-old animals than in cultures from 18-day-old animals. Analysis through the polymerase chain reaction (PCR) indicates that (Bu)2cAMP or FSH treatment increases the levels of the mRNAs for both the potentially soluble form and the transmembrane form of SLF in cultured Sertoli cells. The ratio between mRNAs encoding the potentially soluble form and the transmembrane form of SLF increases during postnatal testis development, and it is higher in cultured Sertoli cells with respect to total testis, suggesting that, under the in vitro conditions, SLF could be produced by Sertoli cells mainly as a soluble factor. Soluble recombinant SLF stimulates, in a dose-dependent fashion, thymidine incorporation in cultures of isolated germ cell populations enriched in the mitotic stages (spermatogonia), independently of the presence of serum, whereas cAMP analogs have no effect. Autoradiographic analysis shows that SLF selectively stimulates DNA synthesis in type A spermatogonia.

Alternative Splicing↗

PubMed1992

Sex-determining region Y (SRY) in a patient with 46,XX true hermaphroditism.

Using a polymerase chain reaction method, a search for Y-specific DNA sequences was made in samples derived from tissues of a 46,XX true hermaphrodite. We found a sequence of SRY in the ovotestis, skin and leukocytes. Other DNA sequences, which covered the pseudoautosomal boundary region, amelogenin gene and DYZ1 locus of Y-chromosome were not detected. The SRY gene detected in the patient by the polymerase chain reaction was not detected by Southern blot analysis, using the SRY fragment as a probe. These findings suggest that in the patient there is a mosaicism of cells with and without part of the Y chromosome, including the SRY sequence. As the SRY sequence was responsible for the development of the gonadal primordium to the ovotestis, SRY seems essential for gonadal differentiation in testis development.

Base Sequence↗

PubMed1995

Thyroid hormone receptor beta mRNA expression in Sertoli cells isolated from prepubertal testis.

A polymerase chain reaction (PCR)-based assay was used to evaluate the expression of thyroid hormone receptor beta mRNA in Sertoli cells isolated from both prepubertal rat and piglet testes. The expression of an mRNA coding for the functional thyroid hormone receptor beta isoform, as established by the PCR assay, agrees with the presence of specific tri-iodothyronine (T3) -binding sites in the Sertoli cell nuclei of both species, as previously evaluated by displacement analysis. The results ratify the existence of a functional T3 receptor in the prepubertal testis and confirm the Sertoli cell as a specific target for thyroid hormone action on the developing testis.

Animals↗

PubMed1991

Cell-cell interactions and the regulation of testis function.

Regulatory interactions have been shown to occur between all the testicular cell types considered. The paracrine factors mediating these interactions generally influence either cellular growth or differentiation. The regulation of cellular growth is essential in the developing testis and is required for the maintenance of spermatogenesis in the adult testis. The rapid rate of germinal cell proliferation and the continuous but slowed growth of the peritubular cells and Leydig cells requires the presence of specific growth factors in the adult. Therefore, cell-cell interactions have evolved that involve growth factors such as IGF, TGF-alpha, TGF-beta and NGF. Other growth factors such as FGF or less characterized components like the seminiferous growth factor (SGF) also may be involved in the paracrine regulation of testis cell growth. An alternate cellular parameter to cell growth to consider is the regulation of cellular function and differentiation. A number of endocrine agents and locally produced paracrine factors have been shown to control and maintain testis cell function and differentiation. Cell-cell interactions mediated by factors such as androgens, POMC peptides, and PModS are all primarily directed at the regulation of cellular differentiation. Therefore, the agents which mediate cell-cell interactions in the testis can generally be categorized into factors that regulate cell growth or those which influence cellular differentiation. The specific cell-cell interactions identified will likely be the first of a large number of cellular interactions yet to be investigated. Although a number of potentially important cell-cell interactions have been identified, future research will require the elucidation of the in vivo physiological significance of these interactions. The existence of different cell types and potential cell-cell interactions in a tissue implies that the actions of an endocrine agent on a tissue will not simply involve a single hormone and single cell. The endocrine regulation of testis function will have effects on cell-cell interactions and be affected by local cell-cell interactions. The ability of LH to influence Leydig cell androgen production promotes a cascade of interactions mediated through several cell types to maintain the process of spermatogenesis. FSH actions on Sertoli cells also promote cell-cell interactions that influence germinal cell development, peritubular myoid cell differentiation and Leydig cell function. Therefore, elucidation of the endocrine regulation of testis function requires an understanding of the local cell-cell interactions in the testis.

Animals↗

PubMed1996

Three daily melatonin infusions alter gonadal development but not GnRH neuron number in the medial preoptic area or diagonal band of Broca in Siberian hamsters.

Among juvenile Siberian hamsters reproductive development is associated with an increased number of unipolar GnRH-immunoreactive neurons in the diagonal band of Broca and medial preoptic area. In the present study, GnRH neuron morphology was assessed in male juvenile hamsters which were treated with three daily melatonin (MEL) infusions to initiate or delay gonadal development. Hamsters gestated in short days were transferred to constant light (LL) at day 14 of age and infused on days 18-20 with MEL for 6 h/day. This treatment stimulated testis development compared to that in saline-infused controls. By contrast, testis growth was suppressed by three 12 h MEL infusions in long-day gestated hamsters in LL from day 14. The number of unipolar and bipolar GnRH neuron subtypes was the same irrespective of MEL infusion or preweaning photoperiod. Thus, gonadal response to three MEL infusions is independent of changes in GnRH neuron number.

Animals↗