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PubMed2004

The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis.

One of the most promising applications of microarrays is the study of changes in gene expression associated with the growth and development of mammalian tissues. The testis provides an excellent model to determine the ability of microarrays to effectively characterize the changes in gene expression as an organ develops from birth to adulthood. To this end, a developmental testis gene expression time course profiling the expression patterns of approximately 36 000 transcripts on the Affymetrix MGU74v2 GeneChip platform at 11 distinct time points was created to gain a greater understanding of the molecular changes necessary for and elicited by the development of the testis. Additionally, gene expression profiles of isolated testicular cell types were created that can aid in the further characterization of the specific functional actions of each cell type in the testis. Statistical analysis of the data revealed 11 252 transcripts (9846 unique) expressed differentially in a significant manner. Subsequent cluster analysis produced five distinct expressional patterns within the time course. These patterns of expression are present at distinct chronological periods during testis development and often share similarities with cell-specific expression profiles. Analysis of cell-specific expression patterns produced unique and characteristic groups of transcripts that provide greater insight into the activities, biological and chronological, of testicular cell types during the progression of spermatogenesis. Further analysis of this time course can provide a distinct and more definitive view into the genes implicated, known and unknown, in the maturation, maintenance, and function of the testis and the integrated process of spermatogenesis.

Animals↗
PubMed2004

A dominant-negative isoform of hypoxia-inducible factor-1 alpha specifically expressed in human testis.

Spermatogenesis in the seminiferous tubuli of the testis occurs under a high proliferation rate, suggesting considerable oxygen consumption. Because of the lack of blood vessels, the oxygen partial pressure in the lumen of these tubuli is very low. We previously identified a testis isoform of the hypoxia-inducible factor (HIF)-1alpha in the mouse, termed mHIF-1alphaI.1. Here, we demonstrate that expression of mHIF-1alphaI.1 increases during puberty, further demonstrating its gene induction in postmeiotic germ cells. Using 5'-rapid amplification of cDNA ends, we identified a novel HIF-1alpha isoform in the human testis, called hHIF-1alphaTe. Like mHIF-1alphaI.1, hHIF-1alphaTe mRNA is derived from an alternative promoter-first exon combination, but with a different genomic organization and a different nucleotide sequence. Reverse transcription-polymerase chain reaction analysis confirmed that hHIF-1alphaTe is exclusively expressed in the testis. As determined by immunofluorescence of ejaculated sperm cells, HIF-1alpha protein is mainly localized in the postacrosomal head and in the midpiece of spermatozoa. Though overlapping with mitochondrial localization in human and mouse spermatozoa, neither hHIF-1alphaTe nor hHIF-1alpha associated with mitochondria. In contrast with the ubiquitously expressed HIF-1alpha protein and the mouse testis-specific mHIF-1alphaI.1 isoform, the hHIF-1alphaTe mRNA sequence predicts a protein with an N-terminal truncation of the DNA-binding domain. As shown by yeast two-hybrid assays, hHIF-1alphaTe still formed heterodimeric complexes with HIF-1beta. However, hHIF-1alphaTe was incapable of forming a DNA-binding HIF-1 complex. Overexpression of exogenous hHIF-1alphaTe resulted in the inhibition of the endogenous HIF-1 transcriptional activity, demonstrating that the testis-specific hHIF-1alphaTe isoform is a dominant-negative regulator of normal HIF-1 activity.

Aging↗
PubMed1983

Ram rete testis fluid contains a protein (clusterin) which influences cell-cell interactions in vitro.

Ram rete testis fluid is shown to elicit clustering of suspensions of Sertoli cells from testes of immature rats, TM-4 cells derived from mouse testis, and erythrocytes from several species. Details of bioassay procedures and characteristics of the phenomenon are reported. Concanavalin A and wheat germ agglutinin prevent aggregation elicited by rete testis fluid, and this inhibition is specifically prevented by alpha-methylmannoside and N-acetyl-glucosamine, respectively. Influences of rete testis fluid on cell aggregation are not dependent on exogenous calcium, but clustering is blocked by various metabolic inhibitors such as dinitrophenol. Rete testis fluid addition to mixed suspensions of erythrocytes and TM-4 cells is followed by separate aggregation of each cell types. Using aggregation of TM-4 cells suspended in simple medium at low density in rotation as a bioassy, we have determined which fractions in rete testis fluid retain activity. We have shown that a heat-stable, trypsin-sensitive protein, having an isoelectric point below pH 4.0, retains the capacity to aggregate cells. We discuss the possible functions of this protein, named clusterin, in cell interactions.

Animals↗
PubMed1985

Effect of photoperiod and melatonin on testis development and regression in wild European rabbits (Oryctolagus cuniculus).

Testis size in male wild rabbits (Oryctolagus cuniculus) kept in an enclosure in Cambridgeshire, U.K. was maximal during May and June and minimal between October and December. Regression occurred after the summer solstice and recrudescence occurred after the winter solstice. Rabbits kept in long days for 25 wk showed no sign of spontaneous testis regression. Hence, testis regression during the summer is probably not due to development of refractoriness to long days. Testis regression occurred in rabbits transferred from long (16L:8D) to short (8L:16D) days. Within 8 wk of the transfer spontaneous regrowth of the testes occurred and the rabbits moulted, and after 16 wk the testes had recovered to their size before the transfer. Subcutaneous implants of melatonin given to rabbits in long days mimicked the effect of a transfer to short days by causing testis regression followed by recrudescence. Moult occurred in rabbits immediately after short day- or melatonin-induced testis regression. The study demonstrates that seasonal reproduction in male wild rabbits in Britain is largely controlled by changes in photoperiod and that this is probably mediated via the pineal gland.

Animals↗
PubMed1992

Genes regulating testis size.

The studies described here provide information about the genetic and morphological bases for the significant differences in testis size among three closely related C57BL mouse substrains: C57BL/6J, C57BL/6ByJ, and C57BL/10J. C57BL/6J mice have normal-size testes while the other two substrains have small-size testes. Genes controlling testis size are postulated to be among the estimated forty genes that differ between the C57BL/6J and C57BL/6ByJ substrains. The number of genes involved in testis size regulation was examined using recombinant inbred mouse strains. An investigation of the role of Y chromosome genes was performed by completing molecular analyses with a mouse Y chromosome-specific probe. Sertoli and germ cell counts provided insight into the morphological basis for the different testis sizes. The experimental results suggest that there are at least two autosomal testis-size genes and that they control testis size by regulating the number of Sertoli cells.

Animals↗
PubMed1992

The effect of testosterone withdrawal and subsequent germ cell depletion on transferrin and sulfated glycoprotein-2 messenger ribonucleic acid levels in the adult rat testis.

We have examined the effects of decreasing intratesticular testosterone concentration and of decreasing germ cell number on levels of transferrin mRNA and sulfated glycoprotein (SGP)-2 mRNA in the adult rat testis. Intact rats received implants of testosterone- and estradiol-filled capsules to suppress LH secretion from the pituitary, thereby suppressing Leydig cell testosterone production. The levels of intratesticular testosterone declined 70% to 20 ng/ml within 3 days, were reduced further to approximately 15 ng/ml by 14 days, and subsequently reached a minimum of about 10 ng/ml. In contrast, the number of elongated spermatids per testis remained unchanged through 14 days, then declined to fewer than 20% of normal between 14 and 28 days, and reached zero by 56 days postimplantation. Likewise, both pachytene spermatocytes and round spermatids declined only after 14 days postimplantation. Northern blots of testicular RNA showed that Sertoli cell transferrin mRNA per testis decreased markedly between 14 and 28 days postimplantation. However, SGP-2 mRNA per testis was unchanged over the time course of the experiment. The decrease in transferrin mRNA, concomitant with germ cell loss, suggests that this mRNA is regulated by the number of germ cells in the testis and not directly by testosterone. In contrast, the constant level of SGP-2 mRNA in the face of reduced intratesticular testosterone and the subsequent loss of germ cells suggests that this mRNA is constitutively maintained in the adult rat testis.

Animals↗
PubMed1993

Regulated expression of testis angiotensin-converting enzyme during spermatogenesis in mice.

A unique isozyme of angiotensin-converting enzyme (ACE) is produced in large amounts by developing germ cells and is referred to as testis ACE. This protein results from the transcriptional recognition of a sperm-specific promoter during spermatogenesis. The expression within germ cells of testis ACE mRNA was studied using in situ hybridization, ribonuclease (RNase) protection, and Northern analysis. In parallel, testis ACE protein expression was measured through use of Western blot analysis and immunohistochemistry. Although testis ACE protein is first detected in step 10 spermatids, testis ACE mRNA is first detected in a developmentally younger cell population, late pachytene spermatocytes. Thus, testis ACE mRNA is translationally arrested for a period of several days until late in spermiogenesis.

Animals↗
PubMed1994

Leukocyte populations of the adult rat testis following removal of the Leydig cells by treatment with ethane dimethane sulfonate and subcutaneous testosterone implants.

The influence of the Leydig cells on the leukocyte population of the testis was investigated. Leydig cells were destroyed by ethane dimethane sulfonate (EDS) treatment in adult male rats, with or without low-dose s.c. testosterone implants to prevent Leydig cell recovery. Leukocytes were counted in perfusion-fixed frozen testis sections, by use of cell-specific monoclonal antibodies (mAbs) with immunoperoxidase detection, or toluidine blue staining. The majority (81%) of testicular leukocytes (OX1+) were immunopositive for the resident macrophage-specific mAb, ED2, and/or the monocyte/macrophage/dendritic cell mAb, ED1. The remaining leukocytes were principally T lymphocytes (R73+). B lymphocytes (OX33+) and metachromatic mast cells were not observed in the normal testis. Treatment with EDS caused a transient increase in ED1+, ED2+, and R73+ cell numbers in the testis, although other evidence of an inflammatory reaction, such as increases in major histocompatibility complex class II antigen, interleukin-2 receptor expression, or capillary permeability, were not observed. At 21 days after EDS treatment, there was a significant decline in macrophage numbers (to approximately 50% of control testis), and T lymphocytes returned to pretreatment levels. After Leydig cell recovery (41 days after treatment), macrophages also returned to pretreatment levels in EDS-treated rats, but remained reduced in EDS-treated animals with testosterone implants. In addition, EDS treatment stimulated a progressive increase in intertubular mast cells, which was significantly inhibited in the testosterone-implanted rats. The data indicate that numbers of testicular macrophages and mast cells, but not of lymphocytes, within the adult rat testis are directly or indirectly regulated by the Leydig cells.

Animals↗
PubMed1995

Efferent innervation of the rat testis.

Previous assessments of the autonomic nerve supply of the male genital tract concluded that the testis received sympathetic input primarily from paraaortic ganglia, particularly the spermatic ganglion. We challenged this consensus by using retrograde axonal tracing to examine the source and distribution of efferent fibers reaching the testis of adult rats. We also used immunohistochemical methods to assess putative neurotransmitters in testicular neurons of the abdominal and pelvic ganglia. The results indicate the majority of retrogradely labeled cell profiles were localized within the major pelvic (38%) and pelvic accessory ganglia (37%), and only a few labeled cell profiles were present in the paraaortic and spermatic ganglia. Injection of FluoroGold and Fast Blue dyes into the respective right and left testis demonstrated that 17% of the neurons in pelvic ganglia were labeled when tracers were microinjected beneath the capsule of the contralateral testis. About 8% of the neurons were labeled both with FluoroGold and Fast Blue, suggesting that certain neurons can provide simultaneous input to the left and right testicles. Immunohistochemical results showed that tyrosine hydroxylase, a marker for noradrenergic fibers, was present in over 33% of the cell profiles labeled with either FluoroGold or Fast Blue. Some 27% of the fluorescent-labeled cell profiles were positive for neuropeptide Y, while 22% were immunoreactive for vasoactive intestinal polypeptide. No evidence for vasoactive intestinal polypeptide immunoreactivity was detected within the testis, but neuropeptide Y-immunoreactive fibers were present in the tunica albuginea and testicular vasculature. Catecholamine fluorescent fibers were distributed sparsely throughout the periphery of the testis in association with the capsule, vasculature, and interstitium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Abdominal position of the rat testis is associated with high level of lipid peroxidation.

Experimental cryptorchidism in the adult rat induces lipid peroxidation as a sign of oxidative stress. To further elucidate the role of free radicals and antioxidant enzymes in the degeneration of testis in cryptorchidism, we first studied testes of untreated rats before and after the normal testicular descent. In the second experiment, primary unilateral cryptorchidism was induced by surgically attaching one testis of each rat to the abdomen before testicular descent. The level of lipid peroxidation was detected by formation of fluorescent chromolipids and diene conjugates. At the age of testicular descent (18-21 days), the level of fluorescent chromolipids dropped to one third (p < 0.05). Correspondingly, the level of diene conjugates was 69% (p < 0.05) higher at 18 than at 30 days of age. The antioxidant enzyme activities did not change at the time of testicular descent. Primary unilateral cryptorchidism was induced at the age of 13 days. At 25 or 35 days of age, the level of diene conjugates was higher in the cryptorchid testes than in the contralateral scrotal testes (+39%, p < 0.01, and +51%, p < 0.001, respectively). In the abdominal testes, the mRNA of CuZn superoxide dismutase (SOD) was increased by 69% (p < 0.05) at 25 days, whereas by 35 days of age enzymatic CuZn SOD activity was slightly decreased and catalase activity increased. The present results show that the abdominal position of the testis, either before normal testicular descent or in experimental cryptorchidism, is associated with a high level of lipid peroxidation. The data provide evidence that increased production of reactive oxygen species could contribute to degeneration of the cryptorchid testis. The oxidative testis. The oxidative stress in the cryptorchid testis is not explained by inactivation of antioxidant enzymes.

Aging↗
PubMed1996

Functional aromatase expression in porcine adrenal gland and testis.

The expression of aromatase cytochrome P450 (P450arom) in the adrenal glands, testes, and placentas of fetal and newborn pigs was investigated. Western immunoblot analysis detected a single 48-50-kDa protein band in these tissues as well as in other porcine tissues known to express P450arom including Day 12 tubular conceptuses, theca interna, and granulosa. Slight differences in migration suggested that the P450arom protein expressed in the testis was larger than that in the adrenal gland, which was, in turn, larger than that in placenta, theca, and granulosa. Consistent with P450arom expression in these tissues, a cDNA encoding porcine P450arom hybridized to a 2.3-kb transcripts in Northern analyses of porcine blastocysts, placentas, and fetal and newborn adrenal glands and testes, as well as in theca and granulosa tissues from preovulatory follicles. No differences in transcript size were detectable among tissues. The identity of P450arom transcripts was confirmed by sequence analysis of partial cDNA clones amplified from porcine fetal adrenal glands, testes, and placentas according to the RACE procedure. The sequences of the adrenal and testis clones were identical but differed from the placental sequence, which represented the first 85 amino acids of porcine P450arom. Specifically, the adrenal and testis clones expressed transcripts that resembled the ovarian isoform of porcine P450arom, rather than the porcine placental isoform, predicting a two-amino acid deletion and 12 predicted amino acid substitutions. P450arom activity was examined to further define expression in these tissues. Activity in adrenal, testis, and placental homogenates was inhibited by 4-hydroxyandrostenedione (4OH-A4) whereas inhibition by etomidate was demonstrated in the adrenal and testis homogenates but not in the placental homogenates. The sensitivity of activity in the newborn porcine adrenal glands and testes to inhibition by etomidate was similar to that of ovarian P450arom activity. The level of P450arom activity was highest in the placenta and lowest in the adrenal gland, and no effect of fetal sex was noted in either tissue. Immunocytochemical studies localized the expression of P450arom in the adrenal gland of newborns to cells at the corticomedullary junction and, with greater intensity, to cells around the developing medullary lobules. The same cells expressed cytochrome P450 17 alpha-hydroxylase, the expression of which also extended throughout the zona fasciculata. The interstitial cells were the site of P450arom expression in the testis, but no expression could be detected in any cells with the spermatic tubules. These data demonstrate that fetal and newborn porcine adrenal glands and testes express an active P450arom that resembles the isoform expressed in the ovary. The localization of adrenal expression suggests a possible role in medullary maturation and function in the fetal and newborn pig.

Adrenal Glands↗
PubMed1996

Microtubule-associated protein-2 in the rat testis: a novel site of expression.

The testis is one of the most abundant sources of microtubule networks. These networks include mitotic and meiotic spindles, the spermatid manchette and axoneme, and the Sertoli cell cytoskeleton. Microtubules are composed of alpha- and beta-tubulin subunits that are polymerized and stabilized by a variety of microtubule-associated proteins (MAPs). One of these, MAP2, has been extensively characterized as a brain-specific protein with the capacity to bind tubulin, cAMP-dependent kinase, and calmodulin. MAP2 mRNA is processed into at least two variants encoding proteins designated MAP2a, MAP2b, and MAP2c. Of the 5.7 kb of coding sequence in the 9-kb mRNA that encodes MAP2a and MAP2b, a deletion of approximately 4 kb produces mRNA encoding MAP2c, which consists of only the N- and C- terminal regions of MAP2b. To determine whether MAP2 was present in the rat testis, microtubule preparations were isolated from adult rat testis and brain by means of taxol-mediated polymerization and analyzed by gel filtration, ELISA, and Western blotting using polyclonal and monoclonal antibodies reactive with MAP2. A 74-kDa protein corresponding to MAP2c was detected in the testis. These results were confirmed by Northern blot analysis of total RNA from adult rat brain and testis with cDNA probes that distinguish between the known MAP2 splice variants. The predominant mRNAs in testis of 6 kb and 2.5-3.5 kb corresponded to MAP2c. A single 6-kb mRNA with the potential to encode MAP2c was detected in enriched preparations of immature Sertoli cells and adult Leydig cells. Round spermatids contained at least two MAP2 mRNAs between approximately 2.5 and 3.5 kb in size that displayed a stage-specific pattern of expression. Immunohistochemistry showed a MAP2-like protein in both somatic and germ cells, with a particularly distinct localization within the cytoplasm of primary and secondary spermatocytes at stage XIV of the seminiferous cycle during meiotic metaphase. In addition to cytoplasmic staining, a novel localization of this protein was observed in the nucleus of many testicular cells.

Animals↗
PubMed1996

Expression of triosephosphate isomerase transcripts in rat testis: evidence for retinol regulation and a novel germ cell transcript.

Vitamin A is essential for mammalian spermatogenesis. To isolate retinol-induced cDNAs from rat testis, vitamin A-deficient (VAD) rats were treated with retinol for 4 h and mRNA isolated from their testes was used to construct a subtractive cDNA library. One cDNA isolated from this library contained a sequence for triosephosphate isomerase (TPI). Northern blot analysis showed that the TPI cDNA hybridized with two transcripts in testis. A 1.4-kb transcript, which is the sole transcript found in most tissues, was expressed in the somatic cells of testis, whereas a novel 1.5-kb transcript was detected only in haploid spermatids. However, only the level of the shorter transcript increased with retinol treatment of the testis or of cultured Sertoli cells. Furthermore, screening of an adult rat testis cDNA library with the TPI cDNA yielded a cDNA putatively corresponding to the 1.5-kb transcript. Sequence analyses of the two TPI cDNAs revealed a 100-bp deletion in one cDNA that may be due to use of an alternative polyadenylation signal. These results suggest independent processing mechanisms for TPI expression in the somatic cells and the haploid germ cells of testis.

Aging↗
PubMed1996

Testis of prepubertal rhesus monkeys receives a dual catecholaminergic input provided by the extrinsic innervation and an intragonadal source of catecholamines.

The mammalian testis is innervated by extrinsic catecholaminergic nerves and responds to catecholamines with steroid secretion. Although the primate testis has also been shown to be innervated, potential differences in the density of this innervation between immature and sexually developed individuals have not been described. A recent study demonstrated that the primate ovary contains a network of neuron-like cells and that some of these cells are catecholaminergic. It is thus possible that the male gonad is also endowed with a similar intragonadal source of catecholamines. The present study addresses these two issues. Catecholaminergic nerves were identified as such by their content of immunoreactive tyrosine hydroxylase (TH; the rate-limiting step in catecholamine biosynthesis), and in some cases by glyoxylic acid histochemistry. Fibers containing TH were abundant in testes from juvenile animals (1-2 yr of postnatal life), but the density of this innervation was not maintained in adult animals, whose testis showed only a few TH-positive fibers scattered in the interstitial tissue. Testicular norepinephrine (NE) concentration was much lower in adult than in juvenile animals, suggesting that the marked increase in testicular weight that occurs with the attainment of sexual maturity is not accompanied by corresponding changes in NE content. At the ultrastructural level, testicular nerve fibers contained pleiomorphic, dense-core and clear vesicles, suggesting the presence of catecholamines and other neurotransmitters. In addition to this extrinsic catecholaminergic innervation, prepubertal testes, but not adult gonads, contained an intrinsic population of TH-immunopositive neuron-like elements, identified as cells by confocal scanning laser microscopy. To determine whether the prepubertal monkey testis indeed expresses the TH gene, testicular RNA was subjected to reverse transcriptase polymerase chain reaction to amplify the 5' end of TH mRNA, which encodes the regulatory domain of the enzyme. The cDNA that was obtained predicts an amino acid sequence similar, but not identical, to that encoded by the alternatively spliced type 1 TH mRNA form present in the adrenal gland. These results indicate 1) that the primate testis receives a dual catecholaminergic input, one provided by the extrinsic innervation and the other by neuron-like cells located within the gonad itself, and 2) that the influence exerted by both sources on testicular function may be more prominent during the prepubertal period than in adulthood. The presence in the testis of a TH mRNA variant encoding amino acid substitutions in its 5' end suggests that regulation of testicular TH enzyme activity may include a gonad-specific component.

Aging↗
PubMed1998

Immunoexpression of the steroidogenic enzymes 3-beta hydroxysteroid dehydrogenase and 17 alpha-hydroxylase, C17,20 lyase and the receptor for luteinizing hormone (LH) in the fetal rat testis suggests that the onset of Leydig cell steroid production is independent of LH action.

The production of testosterone in the adult testis is mainly regulated by LH. Testosterone is essential for normal development of the male fetus. The regulation of testosterone production in the fetal testis is less clear than in the adult testis, and there are indications that at least the onset of androgen production in the fetal testis takes place in an LH-independent way. The aim of the present study was to compare the onset of synthesis and pattern of expression of LH receptors and two important steroidogenic enzymes, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17alpha-hydroxylase/17,20-lyase (P450c17), in fetal rat gonads. Whole fetuses (13.5 and 14.0 postcoitum (p.c.), testes, and ovaries dissected from fetuses on Days 14.5 p.c. to 20.5 p.c. and testes obtained on Days 3, 5, and 7 postpartum were fixed in Bouin's solution and processed for immunocytochemistry. In all samples of fetal testis, 3beta-HSD was detected on Day 14.5 p.c., and immunoexpression of P450c17 appeared one day later on Day 15.5 p.c. Thereafter, immunoexpression of both enzymes remained intense throughout gestation and postnatally. In contrast, immunoexpression of LH receptors was detectable only from Day 16.5, when a few weakly positive cells were present, but it became more intense one and two days later. Similar to the immunostaining for 3beta-HSD and P450c17, immunostaining for LH receptor remained strong throughout gestation and during the first 7 days of postnatal life. No immunoexpression of any of the three proteins studied was detected in the fetal ovary at any age. These data show very early immunoexpression of 3beta-HSD, which was detected one day before the reported start of steroid production by the fetal Leydig cells. In contrast, immunoexpression of LH receptors was evident only after immunoexpression of both the steroidogenic enzymes studied had became apparent and after the reported start of steroidogenesis, which is consistent with the theory that onset of steroidogenesis in the fetal rat testis is an LH-independent process.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Effects of thyroid and luteinizing hormones on the onset of precursor cell differentiation into leydig progenitor cells in the prepubertal rat testis.

Leydig cells in the adult rat testis differentiate during the neonatal-prepubertal period. However, the stimulus for the initiation of their differentiation is still not clear. In the present study our objectives were to test the effects of thyroid hormone and LH on the initiation of precursor cell differentiation into Leydig cells in the prepubertal rat testis. Four groups of Sprague-Dawley rats were used. All treatments began at postnatal Day 1. Rats in groups I, II, and III received daily s.c. injections of saline (200 microl, controls), triiodothyronine (T(3), 50 microg/kg body weight, hyperthyroid), and LH (ovine LH 10 microg/rat/day), respectively. Rats in group IV were made hypothyroid from postnatal Day 1 by adding 0.1% propylthiouracil (PTU) to their mother's drinking water. Testes of rats were collected at 7, 8, 9, 10, 11, 12, 16, and 21 days of age, fixed in Bouin's solution, and embedded in paraffin for immunocytochemical studies. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and LH receptors (LHR) in testicular interstitial cells (other than the fetal Leydig cells) was observed using the avidin-biotin method. In control rats, out of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for 3beta-HSD, beginning from the postnatal Day 11. However, positive immunolabeling for LHR was first detected in these cells at Day 12, i.e., after acquiring the steroidogenic enzyme activity. In T(3)-treated rats 3beta-HSD positive spindle-shaped cells were first observed at Day 9 (i.e., 2 days earlier than controls), and LHR-positive cells were first observed on Day 11 (2 days later than obtaining 3beta-HSD immunoactivity); they were exclusively the peritubular mesenchymal cells. The 3beta-HSD- and LHR-positive spindle-shaped cells were absent in the testis interstitium of LH-injected rats from Days 7 through 12 but were present at postnatal Day 16. In addition, more fetal Leydig cell clusters and fetal Leydig cells in mitosis were present in LH-treated rats compared to rats in all other treatment groups. Following their first detection, the number of positive cells for each protein continued to increase at each subsequent age in controls, T(3)-, and LH-injected groups. In PTU rats, 3beta-HSD and LHR-positive spindle-shaped cells were absent throughout the experimental period. From these observations, it is possible to suggest the following regarding the developing rat testis interstitium. 1) The precursor cells for the adult generation of Leydig cells in the postnatal rat testis are the peritubular mesenchymal cells. 2) Luteinizing hormone does not initiate the onset of mesenchymal cell differentiation into Leydig cells, instead it delays this process. However, daily LH treatment causes mitosis in fetal Leydig cells and increase in fetal Leydig cell clusters. 3) Thyroid hormone is critical to initiate the onset of mesenchymal cell differentiation into adult Leydig cells.

3-Hydroxysteroid Dehydrogenases↗
PubMed2000

Cell proliferation and hormonal changes during postnatal development of the testis in the pig.

Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.

Animals↗
PubMed1982

Radiation dose and biological effects to mouse testis from sodium 32P-phosphate.

Radiation dose to mouse testis was estimated to be about 1.65 rad per microCi of intravenously injected 32P. This high dose to the organ was due to the incorporation of this isotope into the macromolecules of the testis. Up to 30% of the total testis activity was in DNA molecules. Biologic effects on mouse testis from 32P were determined by testis weight loss and the decrease in the number of sperm heads in the testis. Number of sperm heads reached a minimum of 1.3% of control 36 days after injection of 3.5 microCi/g body weight of 32P. Significant decreases in sperm head counts were observed after as little as 0.2 microCi/g body weight of 32P.

Animals↗