Development of germ cell transplants in mice

Development of spermatogonial transplants was studied by using 5- to 6-wk-old histocompatible mice as cell donors and sterile (W-locus) mice as recipients. Groups of animals transplanted with germ cell suspensions were killed at 10 min, 9 h, 24 h, 1 wk, 1 mo, 2 mo, and 3 mo along with age-matched "start" and "end" W-locus controls. Weight of testes increased significantly at 24 h through 3 mo after germ cell transplantation, suggesting that the infused cells quickly stimulated organ function. Small clones of young spermatocytes were evident at 1 mo and sperm at 2 mo. The percentage of tubular profiles containing active spermatogenesis originating from spermatogonia increased with time (0.8% at 1 mo, 8.9% at 2 mo, and 28.2% at 3 mo). Most transplanted germ cells were eliminated from the seminiferous epithelium through phagocytosis by Sertoli cells that occurred primarily before 1 wk, although some pachytene cells were able to proceed through meiosis by 1 wk. A variety of abnormal features are described that characterize developing spermatogenesis in the transplanted testis. Spermatogenesis improved quantitatively and qualitatively with time although released sperm were frequently engulfed by intratubular macrophages and Sertoli cells. A quantitative analysis of spermatogenesis from transplanted germ cells will serve as a basis for improving spermatogonial transplant efficiency.     

Variations in mitochondrial size and ultrastructure during germ cell development

Size variations and ultrastructural changes in mitochondria of developing germ cells of the female hamster were analyzed. Mitochondria in oogonia of foetus and newborn were elongate with transverse cristae. During pre-dictyate meiotic prophase they became small, rounded, and electron-dense with pleomorphic cristae. These changes were largely reversed when dictyate was reached. Maximum mitochondrial size and complexity of cristae were reached just at the beginning of the phase of rapid oocyte growth, and thereafter declined. As mitochondrial size and number of cristae decreased in the rapidly enlarging oocyte, the ratio of length to width increased, as did electron density of the matrix, until the formation of an antrum within the follicle. After antrum formation, the mitochondria again became more rounded and cristae were seldom seen. An attempt is made to correlate changes of mitochondrial morphology with other events occurring during oogenesis.     

A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice

To directly assess c-myc function in cellular proliferation, differentiation, and embryogenesis, we have used homologous recombination in embryonic stem cells to generate both heterozygous and homozygous c-myc mutant ES cell lines. The mutation is a null allele at the protein level. Mouse chimeras from seven heterozygous cell lines transmitted the mutant allele to their offspring. The analysis of embryos from two clones has shown that the mutation is lethal in homozygotes between 9.5 and 10.5 days of gestation. The embryos are generally smaller and retarded in development compared with their littermates. Pathologic abnormalities include the heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females have reduced fertility owing to embryonic resorption before 9.5 days of gestation in 14% of implanted embryos. c-Myc protein is necessary for embryonic survival beyond 10.5 days of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time.     

Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA at the ends of eukaryotic chromosomes. It has been hypothesized that telomerase activity is necessary for cellular immortalization and that telomerase activity is present in cells of germline origin. The objective of the present study was to determine the level of telomerase activity in the following rat cells: 1) oocytes from follicles at different stages of development, 2) spermatogenic cells, and 3) early embryos. Telomerase activity was quantitated using a recently developed, sensitive polymerase chain reaction-based assay and a human kidney cell line (293) as a standard. Telomerase activity was found in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes. The level of enzyme activity in early antral and preovulatory follicles was comparable to that of the 293 cells, while levels in ovulated oocytes were 50-fold lower. Telomerase activity was present in even lower levels in pachytene spermatocytes and round spermatids, and no telomerase activity was detected in spermatozoa from either the caput or the cauda epididymis. After fertilization, telomerase activity was present in 4-cell embryos. Telomerase activity was also detected in several rat somatic tissues. These data demonstrate that telomerase activity is present in germ cells at several stages of differentiation, with the exception of spermatozoa, and suggest that telomerase activity may be important during meiosis. The high levels of telomerase activity in individual oocytes may serve as a marker for monitoring the effects of hormonal agents, aging, and toxins on oocyte quality.     

Steady-state methadone effect on generalized arousal in male and female mice.

Methadone is widely used in treatment of short-acting opiate addiction. The on-off effects of opioids have been documented to have profound differences from steady-state opioids. The authors hypothesize that opioids play important roles in either generalized arousal (GA) or aversive state of arousal during opioid withdrawal. Both male and female C57BL6 mice received steady-state methadone (SSM) through osmotic pumps at 10 or 20 mg/kg/day, and GA was measured in voluntary motor activity, sensory responsivity, and contextual fear conditioning. SSM did not have any effect on those GA behaviors in either sex. Females had higher activity and less fear conditioning than males. The effects of SSM on stress-responsive orexin gene expression in the lateral hypothalamus (LH) and medial hypothalamus (MH, including perifornical and dorsomedial areas) were measured after the behavioral tests. Females showed significantly lower basal LH (but not MH) orexin mRNA levels than males. A panel of GA stressors increased LH orexin mRNA levels in females only; these increases were blunted by SSM at 20 mg/kg. In summary, SSM had no effect on GA behaviors. In females, SSM blunted the GA stress-induced LH orexin gene expression. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Lumbar motor neuron size and number is affected by age in male F344 rats

Motor neurons in the spinal cord of old rats appear similar in size but less numerous compared with those in mature rats; they also contain a large amount of lipofuscin, the lipid peroxidation by-product whose function is largely unknown. The object of this study was to morphometrically characterize motor neurons found in the L4/L5 lumbar spinal cord of mature (6-month) and old (22-month) rats. Paraformaldehyde-fixed, lumbar spinal cords from six rats at each age were embedded in paraffin, sectioned at 6 microm and stained with 0.1% toluidine blue. The nucleolar diameter and area from a minimum of 34 motor neurons per spinal cord were measured. Motor neuron number was calculated using Abercrombie's (Abercrombie, 1946) formula after correcting for tissue shrinkage. Motor neuron number was decreased with age while the neuronal area increased with age. Nucleolar diameter also increased in old rats. Frequency distributions of motor neuron area revealed unimodal distributions of motor neurons rats of both ages. We suggest that larger nucleolar diameter reflects more metabolically active neurons in old rats while larger neuron area is a reflection of the presence of lipofuscin in old motor neurons.     

A Y-chromosomal effect on blastocyst cell number in mice

Karyotopic and cell number analysis of 3.5 day post coitum preimplantation mouse embryos was used to determine whether XY embryos had more cells than XX embryos at the late morula/early blastocyst stage. This proved to be the case for the CD1 strain (for which it had previously been shown that XY embryos form a blastocoel earlier than XX embryos) and for the MF1 strain. However, this increased cell number was not seen in MF1 embryos carrying an RIII strain Y in place of the MF1 Y. Furthermore, interstrain crosses between CD1 and the MF1,YRIII strain showed that the cell number increase segregated with the CD1 Y but not with the RIII Y. It is concluded that the CD1 and MF1 Y chromosomes carry a factor that accelerates the rate of preimplantation development.     

Autonomous cell death of mouse male germ cells during fetal and postnatal period

OBJECTIVE: To determine the utility of CT-determined main pulmonary artery diameter (MPAD) for predicting pulmonary hypertension (PH) in patients with parenchymal lung disease. DESIGN: Retrospective review of right-heart hemodynamic data and chest CT scans in 45 patients. SETTING: Tertiary-referral teaching hospital and VA hospital. PATIENTS: Between October 1990 and December 1995, 36 patients referred for evaluation of parenchymal lung disease or possible pulmonary vascular disease were found to have PH, as defined by mean pulmonary artery pressure (mPAP) > or =20 mm Hg. Nine control patients (mPAP <20 mm Hg) were also identified (4 from hospital records search, 5 after evaluation for possible PH). RESULTS: CT-determined MPAD was 35+/-6 mm in patients with PH and 27+/-2 mm in control subjects. In our group of patients, MPAD > or =29 mm had a sensitivity of 87%, specificity of 89%, positive predictive value (PPV) of 0.97, and positive likelihood ratio (LR) of 7.91 for predicting PH; in the subgroup of patients with parenchymal lung disease (n=28, PH and control subjects), MPAD > or =29 mm had a sensitivity of 84%, specificity of 75%, PPV of 0.95, and positive LR of 3.36 for predicting PH. The most specific findings for the presence of PH were both MPAD > or =29 mm and segmental artery-to-bronchus ratio > 1:1 in three or four lobes (specificity, 100%). There was no linear correlation between the degree of PH and MPAD (r=0.124). CONCLUSIONS: CT-determined MPAD has excellent diagnostic value for detection of PH in patients with advanced lung disease. Therefore, standard chest CT scans can be used to screen for PH as a cause of exertional limitation in patients with parenchymal lung disease. Because CT is commonly used to evaluate parenchymal lung disease, this information is readily available.     

Mammary tumour induction by pituitary grafting in male mice: an animal model for male breast cancer

Isologous anterior pituitary grafting, 4 each, to 3-4-month-old SHN and SLN male mice resulted in an appearance of mammary tumours from 8 months of age and the incidence at 12 months reached 53.8% in each strain. All tumours were diagnosed as type B adenocarcinomas. In association with the results, normal mammary gland growth and mouse mammary tumour virus (MMTV)-gp52 antigen levels in the submaxillary glands were stimulated by the treatment in these strains. The effect of pituitary grafting was much less in GR/A male mice in which no mammary tumours appeared.     

Intermale fighting affected by home-cage odors of male and female mice.

Conducted 3 experiments to test the hypothesis that male mice can produce an aggression-inhibitory or facilitory odor under varying conditions of social stimulation. In Exp I, 25 male fighter mice fought 25 castrated opponents more vigorously in soiled home cages of either single or stable groups of male mice than they did in clean cages. Fighting was also stimulated in cages briefly occupied by other pairs of fighting mice. It is concluded that the release of aggression-promoting home-cage odors by male mice is not necessarily a consequence of social instability and that they are of urinary, rather than preputial, origin. In Exp II (with 21 fresh Ss) and Exp. III (with 30 Ss from Exp I), odors deposited by either single or groups of female mice greatly reduced fighting, indicating that their urinary aggression-inhibiting pheromone is effective after deposition upon the home environment. Testosterone propionate implants abolished the females' inhibitory pheromone and did so independently of their enlarged preputial glands. (20 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Developmental exposure of male germ cells to 5-azacytidine results in abnormal preimplantation development in rats

Methylation of cytosine residues in mammalian DNA is established during gametogenesis and embryogenesis; it plays an important role in gene regulation and normal embryonic development and has also been implicated in genomic imprinting. In the present study, we evaluated whether paternal administration of 5-azacytidine, a drug that is incorporated into DNA and blocks DNA methylation, could alter male germ cell development and function. A drug that does not block methylation, 6-azacytidine, served as a control. Adult male Sprague-Dawley rats (n = 4-8 per group) were treated i.p., three times per week for 4 and 11 wk, with saline or 2.5 (low dosage) or 5.0 (high dosage) mg/kg of 5-azacytidine and 6-azacytidine. After each of the treatment periods, males were mated to determine effects on fertility and embryo development. Although neither 6-azacytidine nor 4 wk of 5-azacytidine treatment affected male reproductive organ weights or sperm counts, 11 wk of 5-azacytidine resulted in dose-dependent reductions in testis and epididymal weights and sperm counts. Both dosages of 5-azacytidine resulted in significant increases in preimplantation loss, and the high dosage of 5-azacytidine caused a decrease in fertility. Examination of embryos on Day 2 of gestation revealed a striking dose-dependent increase in the average number of abnormal embryos per litter sired by the males treated with 5-azacytidine (saline, 0.33 +/- 0.24; low dosage, 2.64 +/- 0.92; high dosage, 10.09 +/- 0.95). In summary, paternal administration of 5-azacytidine interfered with normal male germ cell development and resulted in alterations in fertilization and early embryo development. We suggest that 5-azacytidine-induced alterations in germ cell DNA methylation patterns may be one of the underlying mechanisms, since similar dosages of the analogue 6-azacytidine had no effect on male reproduction and progeny outcome.     

Bilateral hypothalamic dopamine infusion in male Zucker rat suppresses feeding due to reduced meal size

PURPOSE: To determine whether vascular, ischemic, and inflammatory causes of bowel wall thickening in children can be differentiated at gray-scale and color Doppler ultrasonography (US). MATERIALS AND METHODS: Thirty-seven children with acute bowel disease underwent graded compression US. Findings of bowel wall thickness, wall echotexture, location of bowel involvement, and presence of color Doppler flow were evaluated. Diagnoses were classified as inflammation (n = 25), vasculitis (n = 7), or ischemia (n = 5) and were confirmed with findings from colonoscopy and biopsy, stool culture analysis, surgery, and cutaneous biopsy, and with a combination of clinical and laboratory data. RESULTS: Patient age (P = .0022), bowel wall thickness (P = .0001), and color Doppler flow (P = .0013) were statistically significantly related to disease type. Wall thickening and absence of visible color Doppler flow suggested ischemia. Older patient age and visible color Doppler flow suggested inflammation, whereas younger patient age and visible color flow suggested vasculitis. Difference in location of bowel disease in patients with ischemic versus those with vascular wall thickening was statistically significant (P = .0185). No difference was found between disease type and wall stratification. CONCLUSION: Gray-scale and color Doppler flow US can aid in differentiating ischemic, vascular, and inflammatory bowel wall thickening.     

Introduction: the male germ cell; origin, migration, proliferation and differentiation

Based upon the observation that estrogen acts in the striatum to rapidly modulate dopamine (DA) neural transmission and DA-mediated behaviors, it has been postulated that these effects of estrogen are mediated by a specific, membrane-bound receptor mechanism. To further characterize the pharmacological specificity of the estrogen binding site, the present experiments examine effects of various estrogen agonists on amphetamine (AMPH)-induced DA release from striatal tissue of ovariectomized female rats, using a superfusion method. Catechol estrogens 4-, and 2-hydroxyestradiol, but not 2-methoxyestradiol, significantly enhance AMPH-induced striatal DA release. Estrogen metabolites, estrone and estriol, and the non-steroidal estrogen analog, diethylstilbestrol, are without effects. Estradiol conjugated to bovine serum albumin (BSA) mimics the effect of estradiol to enhance stimulated striatal DA release. These results indicate that the steroidal configuration and hydroxylation on the A-ring of estrogenic compounds may be important determinants of ligand binding to the putative estrogen binding site in the striatum. Furthermore, the effectiveness of the estradiol conjugated to BSA reinforces the idea of an external membrane-bound receptor binding site in the striatum.     

MK-801 and male odours induce c-fos expression in the AOB of juvenile female mice

The odours of adult males, which accelerate the timing of puberty of female mice, activate c-fos in the accessory olfactory bulb (AOB). To test the hypothesis that NMDA receptors are involved in the male odour-induced increase in c-fos expression, we studied the effects of the non-competitive NMDA receptor agonist MK-801 on male odour-induced c-fos expression in the AOB of juvenile female mice. Surprisingly, MK-801 increased FOS-like immunoreactivity (FLI) within the AOB in the absence of male odour and had no effect on male odour-induced c-fos expression. We suggest that MK-801 increases AOB mitral cell activity by disinhibiting GABAergic granule cells, resulting in increased c-fos expression throughout the AOB.