Normal Mouse Testis Research Articles

An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis.

Transgenic mice expressing high levels of the BclxL or Bcl2 proteins in the male germinal cells show a highly abnormal adult spermatogenesis accompanied by sterility. This appears to result from the prevention of an early and massive wave of apoptosis in the testis, which occurs among germinal cells during the first round of spermatogenesis. In contrast, sporadic apoptosis among spermatogonia, which occurs in normal adult testis, is not prevented in adult transgenic mice. The physiological early apoptotic wave in the testis is coincident, in timing and localization, with a temporary high expression of the apoptosis-promoting protein Bax, which disappears at sexual maturity. The critical role played by the intracellular balance, probably hormonally controlled, of the BclxL and Bax proteins (Bcl2 is apparently not expressed in normal mouse testis) in this early apoptotic wave is shown by the occurrence of a comparable testicular syndrome in mice defective in the bax gene. The apoptotic wave appears necessary for normal mature spermatogenesis to develop, probably because it maintains a critical cell number ratio between some germinal cell stages and Sertoli cells, whose normal functions and differentiation involve an elaborate network of communication.

Localization of Heat Shock Proteins in Mouse Male Germ Cells: An Immunoelectron Microscopical Study

We have examined the intracellular distribution of heat shock proteins HSP27 and HSP90 by means of specific antibodies and ultrastructural immunocytochemistry in the normal mouse testis as well as after heat shock. In the unstressed testis, these heat shock proteins are present in the cytoplasm and to a lesser extent in the nucleus throughout spermatogenesis. They do not show preferential association with any specific cytoplasmic structures and are absent from mitochondria. They disappear from the cell nucleus at the stage of elongating spermatids. After heat shock (42°C), both HSP90 and HSP27 increase and partly relocate to the nucleus. Similarly to the localization in unstressed cells, they are mainly associated with perichromatin fibrils and the nucleolus. Moreover, a remarkable increase in the frequency of perichromatin fibrils in Sertoli cells, spermatogonia, and primary spermatocytes is also observed upon heat shock. Finally, a sharp increase in the labeling of HSPs in chromatoid bodies of round spermatids occurs following hyperthermic treatment. Interestingly, these two HSPs are localized on nuclear structures which are actively involved in RNA synthesis and processing, suggesting that they may have protective functions in these processes in a tissue which is particularly sensitive to heat stresses.

CD28–CD80/CD86 Interactions in testicular immunoregulation

The expression of two accessory molecules on antigen-presenting cells (APC), the CD80/ B7-1 and CD86/B7-2 antigens, was studied in the testis of normal and non-obese diabetic (NOD) mice. In addition, the effect of CD28 stimulation on suppression of lymphocytes by testicular products was investigated. The testes of 4-week old NOD mice or normal BALB/c mice and the testis of 17–21-week old BALB/c mice contained no CD80 or CD86 expressing cells. In contrast, CD80+ and CD86+ cells were present in the testis of 14–22-week old NOD mice. The CD80+ cells and most of the CD86+ cells were CD11b/CD18 negative. There were some CD11b/CD18+ cells that expressed CD86 weakly. The CD80+ and CD86+ cells were often located adjacent to the vessel walls where a leukocyte not expressing CD80 or CD86 had attached to the endothelium. Some CD80+ and CD86+ cells were present among the interstitial cells. The CD80 and CD86 antigens could not be observed in the same cells as judged from stainings in parallel sections. Stimulation of ConA-or anti-CD3ϵ-primed peripheral blood or spleen lymphocytes with anti-CD28 was able significantly to antagonize the growth-inhibitory effect of the M r > 5 K fraction of testis extracts, but could not abolish it with increasing concentrations of testis extract. The results suggest that T lymphocytes can not be activated locally in the testis of BALB/c and young NOD mice because of the absence of the necessary CD28 ligands, CD80 and CD86, from the APCs and because of the suppression of T lymphocytes by the testicular products. In the testis of older diabetic NOD mice lymphocyte activation may occur because the testes of these mice contain CD80+; CD11b/CD18−, CD86+; CD11b/CD18+ and CD86+; CD11b/CD18− cells and therefore, CD28 co-stimulation, which can antagonize the suppressive effect of testis extract, may occur. The possibilities for clonal anergy in testicular immunoregulation are discussed.

Differential expression of clusterin in the testis and epididymis of postnatal and germ cell deficient mice.

Clusterin is found in a wide variety of tissues and is expressed in a number of physiological and pathological contexts. It is expressed constitutively in the adult male reproductive tract, specifically the testes and caput of the epididymis. The gene is also induced during apoptotic cell death in the kidney and hormone-dependent tissues such as the prostate and mammary gland. The mechanisms controlling the expression of clusterin gene expression in these tissues are still unknown, although it has been suggested that interactions between the Sertoli cells and germ cells in the testis, or epithelial cells and germ cells in the epididymis, may be required for clusterin expression. To investigate the importance of germ cells in the induction of clusterin expression in these tissues, we have used in situ hybridization to determine the developmental regulation and germ cell dependence of clusterin expression in the reproductive tract of the normal and germ cell deficient male mice. Clusterin mRNA is present in the supporting cells in the testes of normal mice and in both atrichosis (at) and dominant spotting mutant (Wv) mice, both of which are germ cell deficient. On the other hand, the expression of clusterin in the epididymis appears to be at least partially dependent of the presence of germ cells, because segmental localization of clusterin mRNA within the caput of the epididymis that is seen in normal mice is disrupted in the at and Wv mutants. In these germ cell deficient mutants, the steady-state level of clusterin mRNA, which is repressed in segment 1 and 2 of the adult wild type mouse, is expressed at levels similar to those seen at days 7 and 17 during the development of the normal epididymis.

The follicle-stimulating hormone (FSH) beta- and common alpha-subunits are expressed in mouse testis, as determined in wild-type mice and those transgenic for the FSH beta-subunit/herpes simplex virus thymidine kinase fusion gene.

Testicular expression of the endogenous FSH beta-subunit (FSH beta) and common alpha-subunit (C alpha) genes, as well as a Herpes simplex virus type 1 thymidine kinase (tk) transgene, driven by a 2.3-kilobase fragment of the bovine GSH beta promoter, were studied at messenger RNA and protein level in normal and transgenic mice. A major 3.8-kb species of FSH beta messenger RNA was demonstrated i the normal mouse testis by Northern hybridization. This was longer than the main 1.7-kb FSH beta transcript detected in the pituitary gland. Reverse transcription-polymerase chain reaction, followed by Southern hybridization, demonstrated FSH beta and tk expression in the pituitary gland and gonads of adult normal and transgenic mice, respectively. The C alpha expression was detected by reverse transcription-polymerase chain reaction in the pituitary gland and testis. During development, testicular transcription of the FSH beta and tk genes was initiated simultaneously a few days after birth. Immunocytochemistry of adult testes showed stage-specific positive reaction with FSH beta, C alpha, and tk antisera in the pachytene spermatocytes and type B spermatogonia, but not in Sertoli cells. Positive reaction with these antisera was also seen in the interstitial tissue. These results demonstrate testicular expression of the endogenous FSH subunit genes and confirm that the testicular expression of the FSH beta /tk transgene reflects or its subunits play a paracrine or autocrine role in the regulation of testicular function.

Expression of Protein Kinase C delta Gene in Germ Cells

Purpose To investigate the biological functions of protein kinase C delta (PKC delta) in spermatogenesis. Materials and Methods We examined PKC delta transcript in mouse testis by means of in situ hybridization and Northern blotting. Results In testes of normal mice, signals of PKC delta gene expression were detected specifically at the spermatid stage. The PKC delta gene was weakly expressed in 8-week-old mice and highly expressed by 12 weeks. However, the expression was not detected in testes of germ cell-deficient W/W sup v mice even at 12 weeks. Conclusions Protein kinase C delta gene expression may be controlled by specific developmental processes and PKC delta may play a role in spermatogenesis.

Angiotensin-Converting Enzyme in Murine Testis: Step-Specific Expression of the Germinal Isoform during Spermiogenesis

Angiotensin I-converting enzyme (ACE) is known primarily as an endothelial enzyme that plays a critical role in the regulation of blood pressure. Another, shorter isoform of ACE is abundantly expressed in the testes of sexually mature animals. Using antibodies for immunoperoxidase detection and [35S]-labeled riboprobes for in situ hybridization (ISH), we studied the temporal expression and cell distribution of this germinal isoform of ACE in the testis of normal mice and rats as well as of pubertal and sterile mice. In both murine species, specific testicular ACE mRNA and its gene product are present only after completion of meiosis. Through studying two murine species in which spermatogenesis and spermiogenesis have been accurately described, as well as immature and sterile animals, it could be shown that ACE mRNA and its corresponding protein are first synthesized during the cap phase (steps 4-7). The maximum expression occurred during the acrosome phase (steps 8-12). ACE mRNA is no longer detectable in spermatids beyond step 14, whereas its gene product is expressed until the end of spermatid maturation. Therefore, ACE is exclusively produced in haploid germ cells and belongs to the growing family of proteins whose expression during definite maturation steps of spermiogenesis appears to be correlated with the unique process of germ cell differentiation.

Murine Experimental Autoimmune Orchitis (EAO) Induced by Syngeneic Testicular Germ Cells (TC) Alone: Orchitogenic and Lymphostimulatory Activities of TC from Mice at Various Ages

As part of our continuous investigations on the immune pathogenesis of a new experimental autoimmune orchitis (EAO) model without the use of adjavant in C3H/He mice, the developmental patterns of autoantigenicity of viable syngeneic testicular germ cells (TC) during the postnatal period were investigated by examining the orchitogenic and immunogenic activities of TC, the lymphostimulatory activities of TC (the cytokine release from sensitized spleen cells (SPC) in response to TC), and the immunohistochemical localization of autoantigens in the testes of normal mice at various weeks of age. TC from 6-week-old mice had the capability of inducing EAO (orchitogenicity) for the first time. Delayed-type hypersensitivity-inducing capacity and antiTC antibody-eliciting capacity were initially observed in mice immunized with TC from 4-week-old mice. A significant stimulation of interleukin 5 (IL-5) and interleukin 6 (IL-6) production by sensitized SPC was detectable when the TC from 3-week or older mice were employed as a stimulant. Interleukin 2 (IL-2) and interferon-γ (IFN-γ) production was detected with TC from 4-week or older mice. Immunohistochemical staining reaction with anti-TC antisera was primarily localized at the acrosomal portion of spermatids and spermatozoa in the seminiferous tubules and was detected in early and late spermatids of mice as early as 3 weeks. Thus, from these chronological data it is suggested that the appearance of the lymphostimulatory activities of TC consistently precedes that of the orchitogenic activity and that the production of IL-5 and IL-6 (Th 2 cytokine) by sensitized SPC upon in vitro TC stimulation is detectable earlier than the production of IL-2 and IFN-γ (Th 1 cytokine) in the postnatal period.

The murine Niemann-Pick type C lesion affects testosterone production.

We have determined the effects of the Niemann-Pick type C (NPC) lesion, which impairs transport of cholesterol from lysosomes, on the androgenic status of male NPC mice. The mice have low serum testosterone levels resulting from decreased testosterone secretion. Testosterone secretion is reduced in NPC mouse testes incubated with 8-bromo-cAMP, 20 alpha-hydroxycholesterol, and pregnenolone compared to testosterone release by normal mouse testes under identical conditions. Ultrastructural examination of testes revealed a paucity of lipid droplets, extensive accumulation of inclusion bodies, and distorted endoplasmic reticulum in Leydig cells of adult NPC mice. The hypoandrogenemia caused systemic deficiencies in NPC mice. Seminal vesicles, a testosterone-responsive tissue, were underdeveloped in NPC male mice. The testosterone-responsive kidney beta-glucuronidase activity was also underexpressed. Seminal vesicle mass and beta-glucuronidase activity were increased by testosterone treatment of NPC mice. Many hepatic proteins, identified by microsequencing, were also deficient in NPC male mice. Levels of alpha 2-mu-globulin, glutathione S-transferase-pi, carbonic anhydrase-III, and selenium-binding protein increased in normal male mice during puberty, but did not increase in the NPC male mice. Based on the increases in protein expression during puberty, differential expression in males and females, and the reported involvement of androgens in regulating expression of some of these proteins, deficient expression of most of these proteins in male NPC mice appears to result from low testosterone levels. We conclude that a defect in testicular testosterone production in NPC male mice causes a pleiotropic deficiency in androgen-sensitive expression of proteins in various organs.

Increased densities of binding sites for the peripheral-type benzodiazepine receptor ligand [3H]PK 11195 in congenital ornithine transcarbamylase-deficient sparse fur mouse.

Peripheral-type (mitochondrial) benzodiazepine receptors (PTBR) were studied in the brain and peripheral organs (kidney, liver, and testis) of normal male mice (CD-1/Y) and the congenitally hyperammonemic sparse fur (spf/Y) mouse. Radioligand binding assays were performed with [3H]PK 11195, a ligand with high selectivity and affinity for PTBR. Densities (maximal number of binding sites) of [3H]PK 11195 binding sites were greatest in kidney, followed by liver, testis, and brain. Densities of [3H]PK 11195 binding sites were significantly increased in all tissues of spf mice compared with control animals. In view of the localization of PTBR on the outer mitochondrial membrane, changes in PTBR in spf mouse tissues may modulate the altered mitochondrial function and oxidative metabolism, in brain and peripheral tissues, in congenital OTC deficiency. The positron emission tomography ligand 11C-PK 11195 could find an application in the assessment of end organ dysfunction in this disorder.

Suppressed expression of the cytochrome P45017α protein in the testicular feminized (Tfm) mouse testes

The testes of testicular feminized (Tfm) mice synthesize and secrete abnormally low amounts of testosterone, as a consequence of selectively decreased cytochrome P450(17 alpha) activity. To investigate the mechanism of this deficiency, three steroidogenic enzymes were immunolabeled in the testes of normal and Tfm adult (2.5-6 month old) mice. Cholesterol side-chain cleavage cytochrome P450 (P450scc) and delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were detected in the Leydig cells of both normal and Tfm mice whereas, in contrast to normal mice, only a small proportion of Leydig cells were immunostained for cytochrome P450-17 alpha-hydroxylase, C17-->20 lyase (P450(17 alpha)) in the testes of Tfm mice. The numbers of cells differed from male to male and interestingly were markedly higher in the right testis. Explants of testes from Tfm mice were kept in organ culture at 32 degrees C for 45 h, with or without dibutyryl cyclic AMP (100 or 500 mumol/l). All Leydig cells remained positive for P450scc and 3 beta-HSD, and P450(17 alpha) became detectable in the majority of Leydig cells in both left and right testes, showing that the lack of expression of P450(17 alpha) protein in Tfm mouse testes in vivo is not structural but is a regulatory phenomenon.

Unique expression pattern of protein kinase C-θ: high mRNA levels in normal mouse testes and in T-lymphocytic cells and neoplasms

A 2.2-kb cDNA that contains the entire coding region of mouse protein kinase C-θ (PKC-θ) was cloned from skeletal muscle mRNA using reverse transcription and the polymerase chain reaction (PCR). This clone was used as a probe to study the expression of this PKC isoform in normal and transformed hemopoietic cells and other normal tissues. By far the highest steady-state level of PKC-0 MRNA was found as a 2.8-kb transcript on a Northern blot of poly(A) + RNA from testes. High levels were also found in skeletal muscle, spleen, T lymphomas and purified normal T lymphocytes, but these tissues and cells expressed two transcripts, 3.3 kb and 3.8 kb. Lower levels of similar size transcripts were found in normal brain, B lymphocytes and B-lymphocytic tumors and cell lines.

Co‐amplification of c‐myc/pvt‐1 in immortalized mouse b‐lymphocytic cell lines results in a novel pvt‐1/aj‐l transcript

In a series of mouse pvt-1 cDNA clones prepared from an immortalized B-cell line that contains an amplified c-myc/pvt-1 region, we have identified a unique cDNA, AJ-I, which contains 57 bp of pvt-1 sequence (pvt-1a) spliced to a novel sequence of 2.1 kb. We report here that this 3' segment (termed AJ-IX) maps more than 60 kb telomeric to pvt-1a and is encoded by a novel locus (AJ-I) on mouse chromosome 15. The AJ-IX probe detects a transcript that is expressed only in normal mouse brain and testis. Several mast-cell tumors, Ly-I+ B-lymphocytic cell lines and a neuroblastoma also display abundant levels of AJ-IX-specific mRNA. However, splicing of pvt-1a to AJ-IX is found exclusively in Ly-I+ B-cell lines that contain amplified c-myc/pvt-1. We conclude that some features in the generation of this amplicon facilitates the synthesis of a pvt-1/AJ-IX chimeric mRNA that may play a role in immortalization of these Ly-I+ B-cell lines.

Ethylnitrosourea treatment increases lectin binding to mouse germ cells

Germ cell toxicity was assessed by investigating the binding of FITC-labeled lectins to mouse testis cells before and 18 days after treatment with ethylnitrosourea (ENU). Flow cytometry of testis cells dual-labeled with FITC-lectin plus the DNA stain, propidium iodide, allowed analysis of haploid (1C), diploid (2C), and dividing (4C) cell populations. Soybean agglutinin, wheat germ agglutinin, concanavalin A and Limax flavus agglutinin bound to normal mouse testis cells containing 1C, 2C or 4C DNA. Asparagus pea lectin and Bandeireae simplicifolia I isolectin B 4 did not. ENU treatment reduced the number of testis cells and increased lectin binding, particularly of those lectins which bound to untreated cells.

High activity of 17α-oxidoreductase in neonatally grafted mouse testes in adult female mice

Male and female (WB-C57BL/6)F 1 hybrid mice were used. Two testes from neonatal mice were grafted into the spleen of adult male and female mice, and the grafted testes were removed 30 and 60 days after grafting. Normal testes from 30- and 60-day old mice were also used. Testicular homogenates were incubated with [ 14C]4-androstene-3,17-dione or [ 3H]progesterone, and enzyme activities per g wet tissue and progesterone metabolism were examined. Activity of 17α-oxidoreductase in the grafted testes in females (20 nmol/g/h) was approx. 10 times the activity in the grafted testes in males or in the normal testes, whereas 17β-oxidoreductase activity in the grafted testes in females was the lowest among these testes. The bilateral ovariectomy performed 1 month before the grafting of neonatal testes, artificial cryptorchidism performed at 20 days of age, and estrogen treatment for 10 days by diethylstilbestrol pellets resulted in no significant changes in 17α-oxidoreductase activities in 30- or 60-day old grafted, cryptorchid or normal testes. The major 17-hydroxy-C 19-steroids formed in vitro from progesterone by the grafted testes in female mice were testosterone and 17α-hydroxy-4-androsten-3-one (epitestosterone), but the formation of epitestosterone was insignificant in the normal testes. The present results demonstrate for the first time that epitestosterone is formed as one of major C 19-steroids in neonatally grafted mouse testes in females but not in those in males or in normal mouse testes. However, the mechanisms remain unexplained.

Morphometric analysis of testicular Leydig cells in normal adult mice.

Stereological analysis was carried out on Leydig cells in perfusion-fixed testes of normal adult mice. In a decapsulated testis, the seminiferous tubules occupy 89.3% and the interstitial tissue makes up 10.7% of the volume of the testis parenchyma. The Leydig cells comprise 3.8% of testicular volume. There are 24.9 million Leydig cells per cm3 (or gm) of tissue. An average Leydig cell has a volume of 1,533 microns 3 and a surface area of 1150 microns 2. The smooth endoplasmic reticulum (SER) is the most prominent organelle in the Leydig cells, and has a membrane surface area of 2,428 cm2 per cm3 of fresh testis tissue, which is 8.5 times the surface area of the plasma membrane and constitutes 56.9% of the total membranes in Leydig cells. Mitochondria occupy 10.1% of the Leydig cell volume or 11.4% of cytoplasmic volume. The inner mitochondrial membrane (including tubular or vesicular cristae) provides a surface area of about 2855 microns 2/cell and is 2.26 times that of the outer membrane. There are approximately 712 cm2 of inner membranes per cm3 tissue. Mouse Leydig cells have numerous lipid droplets, which average 147 per cell and occupy 5.1% of the cell volume.

Spermatogenesis in the immature mouse proceeds faster than in the adult.

The first appearance of spermatogenic cell types related to the age of the animal was studied in sections and tubular whole mounts of testes of normal mice (Cpb-N strain) up to 34 days pp. The first intermediate spermatogonia and leptotene spermatocytes were seen at days 4 and 7 p.p., respectively. It was found that the subsequent types of spermatogenic cells appear earlier than could be expected if spermatogenesis was to proceed at adult speed. [3H]thymidine labelling studies revealed that within a given interval of time, spermatocytes and spermatids in immature mice develop into more advanced cell types than in adults. The labelling studies and the observation that the cellular associations are always identical to those in the adult, indicate that the rate of acceleration in young mice is the same for spermatogonia, spermatocytes and spermatids. The mean duration of the cycle of the seminiferous epithelium during the age interval of 10 to 30 days p.p. is 7.51 +/- 0.10 days, compared to 8.61 +/- 0.08 in the adult. It increases gradually towards the adult level, reaching the value of 8.45 +/- 0.17 days between days 33 and 56 p.p.

Elevated cholesterol and dolichol synthesis in mouse pachytene spermatocytes.

Results are presented here which demonstrate that the rates of [14C]acetate incorporation into cholesterol and dolichol increased 4- to 5-fold as mouse spermatocytes matured from the preleptotene to prepuberal pachytene stages. The rate of acetate incorporation into cholesterol then decreased in late pachynema, remained low at all subsequent stages of meiosis, and was very low in mature sperm. In contrast, the rate of acetate incorporation into dolichol remained elevated in late pachytene spermatocytes and round spermatids, then decreased and remained low in mature sperm. The ratio of the rate of [14C]acetate incorporation into dolichol to the rate of incorporation into cholesterol increased during late meiotic prophase and remained high in round spermatids; this altered ratio is further evidence of independent regulation of dolichol and cholesterol synthesis in testes. It was shown previously that normal adult mouse testes incorporated acetate into dolichol at a much higher rate (1.8 to 2.4% of the rate of incorporation into cholesterol) than did testes from sterile W/Wv mice (0.02%) or X-irradiated mice (0.24%). This high rate of acetate incorporation into dolichol in adult testes is now attributed to differentiating spermatocytes, with particularly high rates being observed during pachynema.

Effect of testicular extracts on proliferation of spermatogonia in the mouse.

Two intraperitoneal injections with an interval of 4 h between them, of rat testicular extract into adult male mice causes a decrease in the production of A spermatogonia in the compartment of undifferentiated A (As, Apr and Aal) spermatogonia. A significant decrease in the total number of A spermatogonia in stages VII and VIII of the cycle of the seminiferous epithelium was found at 2, 4 and especially 5, 7 and 8 days after treatment. Extracts of rat liver and rat spleen were without effect. In addition, an extract of rat testis containing very few spermatogonia had no effect. It was concluded that the active substance in the extract is synthesized and/or specifically accumulated in the spermatogonial compartment of the testis. Thus the active substance is tissue-specific but not species-specific, since extracts of both rat and bull testes were effective after injection into mice. It is inferred from the data that the effect of injection of testicular extracts is unlikely to be due to cytotoxicity, hormonal changes in the tubular environment or to an immunologic reaction, but is probably due to a spermatogonial chalone. This chalone partially inhibits proliferation of early type A spermatogonia in the normal mouse testis.

Transplantation of the gonads of hypogonadal (hpg) mice.

Testes from hypogonadal (hpg) mice transplanted under the tunica albuginea of the testes of normal mice displayed full spermatogenic activity after 84 days. When ovaries of hpg mice were transplanted to the periovarian capsule of ovariectomized normal females ripe follicles and corpora lutea developed. Although small remnants of normal ovary were found after this operation, the fact that 5 out of 11 normal females bearing transplanted ovaries and mating with heterozygous males raised litters containing hpg mice shows that the ovary of the mutant is capable of producing ova which can be fertilized.