Germ cell removal after induction of cryptorchidism in adult rats.

Cryptorchidism is associated with male infertility due to germ cell loss in response to elevated temperature. However, there is a great deal of contradictory information prevalent on the status of germ cells and their process of removal in the cryptorchid testis. In the present study, we investigate the cell removal from cryptorchid rat testis by the methods of morphology and stereology. The testis weight is reduced according to previous reports after surgical induction of cryptorchidism. Interestingly, the epididymal weight is significantly increased in 7 days after surgery, and the caput epididymis tubules show filling with countless round germ cells. We found that the elongating spermatids (steps 10-13), newborn spermatids (step 1) and the dividing spermatocytes are the most susceptible cells to elevated temperature, and are the first disappeared cells from the seminiferous tubules after surgery. Germ cell removal followed the order, starting first with elongating spermatids and newborn spermatids, followed by round spermatids and elongated spermatids and later extending to spermatocytes.

Drug synergistic antifertility effect of combined administration of low-dose gossypol with steroid hormones in rats.

BACKGROUND: Our previous studies suggested that low-dose gossypol combined with steroid hormones has a reversible antifertility role in adult male rats, and the course of treatment was shorter than that of either gossypol or steroid hormones alone. This result suggested that low-dose gossypol and steroid hormones have a drug synergistic effect on antifertility. The aim of the study was to find the target organs of the antifertility synergistic effect of the combined regimen. METHODS: Thirty-two adult male rats were divided into four groups randomly: group GH, rats were fed orally with gossypol acetic acid (GA, 12.5 mg×kg(-1)×d(-1)) and desogestrel (DSG, 0.125 mg×kg(-1)×d(-1))/ethinylestradiol (EE, 0.025 mg×kg(-1)×d(-1))/testosterone undecanoate (TU, 100 mg×kg(-1)×d(-1)); group G, a single dose of GA (12.5 mg×kg(-1)×d(-1)) was given; group H, the same dosage of DSG/EE/TU as in group GH were administered; group C, rats were treated with vehicle (1% methyl cellulose) as control. Testes and epididymis were removed at 8 weeks post-treatment for evaluating their weight, volumes, volume fraction, and total volume of testicular tissue structures and the seminiferous tubule diameter using stereological assay. Sperm cell numbers and the motility of epididymal sperm were quantitated by flow cytometry and morphological methods. RESULTS: Compared with group C, spermatogenesis was normal in group G and suppressed in groups H and GH. Similar changes of testicular tissue structures and sperm number were found in groups H and GH. The decreases of epididymal sperm number and motility in group GH were greater than that of the low-dose gossypol or steroid hormones alone group. CONCLUSIONS: The suppression of spermatogenesis was induced by steroid hormones in the combined regimen, and the epididymis was the target organ of low-dose gossypol. Combined use of low-dose gossypol and steroid hormones played a comprehensive antifertility role in their synergistic effect on reducing the number and motility of epididymal sperm.

Expression of Hsf1, Hsf2, and Phlda1 in cells undergoing cryptorchid-induced apoptosis in rat testes.

Surgery-induced cryptorchidism, in which the testes are prevented from descending into the scrotal sac, results in testicular germ cell death, and it is commonly used as an experimental tool in the study of spermatogenesis. However, the molecular events underlying the activation of germ cell death remain poorly understood. In the present study, we investigate selective cell loss from cryptorchid rat testis by using DNA flow cytometry and by determining protein and mRNA expression of Hsf1, Hsf2, and Phlda1. The hypo-haploid cell fraction is significantly decreased as early as 3 days after surgical induction of cryptorchidism (from 42.01 ± 5.74% to 15.98 ± 3.88%), followed by a significant decrease in the haploid cell fraction at Day 7. At the latter time point, an apoptotic peak of spermatocytes appears in DNA histograms just before the tetraploid peak; the percentage of aneuploid cells between diploid and tetraploid rises as high as 14.05 ± 2.98% of the total cells in 7-day cryptorchid testis, suggesting that a large number of spermatocytes are undergoing apoptosis. The expression of Phlda1 mRNA is significantly elevated 3 days after induction of cryptorchidism. After 7 days of cryptorchidism, Hsf1 and Phlda1 are strongly expressed in the nucleus and cytoplasm, respectively, of primary spermatocytes. Numerous apoptotic spermatocytes are also observed at this time point. These results suggest that the Hsf1/Phlda1 pathway plays an important role in the apoptosis of primary spermatocytes in cryptorchid testis. We present evidence suggesting that Hsf2 is also involved in germ cell removal in cryptorchid testis.

Stereological measurement of rat's seminiferous tubule.

BACKGROUND: The morphological measurements of seminiferous tubules are important in the studies of testis tissues. The purpose of this study was to evaluate the feasibility of using a stereological method to measure the geometric parameters of seminiferous tubule and to optimize the method. METHODS: A stereological image processing program was developed with Delphi for the stereological measurement. Fields of view were obtained from 15 healthy Wistar rats' testis tissues (n = 247). The diameter, area and volume of seminiferous tubule were estimated with the image processing program by two individual observers. The area results were compared with those obtained by the standard morphometric method of planimetry. RESULTS: Diameter measurements showed the diameters of different seminiferous tubules were almost the same and the mean value of about 50 tubules could be a good representation of the whole structure. Area measurements indicated there was no significant difference between stereology and planimetry (P > 0.05). But the stereological method required about 45% less time. Volume measurement showed the inter-observer variability was small (P > 0.05) and the reproducibility of the stereological method was good. CONCLUSION: The stereological technique was practical and efficient in the quantitative measurement of the rat's seminiferous tubule.

A combined regimen of gossypol plus methyltestosterone and ethinylestradiol as a contraceptive induces germ cell apoptosis and expression of its related genes in rats.

Attempts to develop gossypol and steroidal hormones alone as a male contraceptive have been tested for many years; however, both caused undesirable side effects that have prevented their acceptance. In this study, we formulated a regimen of combined gossypol at a low dose of 12 mg/kg or a high dose of 50 mg/kg plus methyltestosterone 20 mg/kg and ethinylestradiol 100 g/kg daily (12 mg G+H and 50 mg G+H) administered for 6 weeks in adult rats. The possible roles of germ cell apoptosis and related genes expression were studied by techniques of TdT-mediated dUTP nick end-labeling (TUNEL), agarose gel electrophoresis of low-molecular-weight DNA, in situ hybridization and reverse transcription-polymerase chain reaction detection. Results showed that germ cell apoptosis and related genes expression were significantly induced after combined drug administration. The apoptosis index increased 3.86- and 9.65-fold in the 12-mg and 50-mg G+H-treated groups, respectively, as compared to the control group. DNA ladder formation on the agarose gel further validated the findings of TUNEL-stained apoptotic cells. The apoptosis-related genes fas mRNA expression levels increased 0.44- and 1.39-fold, bax mRNA 0.74- and 2.56-fold, caspase-3 mRNA 0.60- and 1.29-fold, and caspase-9 mRNA 2.50- and 4.08-fold, respectively, in the 12-mg and 50-mg G+H-treated groups vs. the control group. These results indicated that our drug regimen applied as a contraceptive could induce rat germ cell apoptosis. The apoptotic process involved fas system, bax and caspase family genes and the apoptotic extent and cell types were gossypol dose-dependent.

[Expression of type II collagen gene and structural change in bone tissues of rats with experimental fluorosis].

OBJECTIVE: To investigate the effects of excessive intake of fluoride on the expression of type II collagen gene and types and morphological change of collagen fiber in the bone tissues of rats. METHODS: A rat model with fluorosis was established by adding 221 mg/L of sodium fluoride (NaF) to drinking water for the rats for 15 days, 30 days and two months, respectively. Type II collagen alpha1 (II) cDNA probe was prepared, and cDNA-mRNA in-situ hybridization was employed to detect change in expression of type II collagen mRNA in the bone tissues of rats with excessive intake of fluoride (221 mg/L NaF). Picrosirius-polarization method was used to observe types of collagen and morphology of collagen fiber in the bone tissues. RESULTS: Chondroblasts were found in the femur and other bone tissues of the rats after exposure to fluoride. cDNA-mRNA in-situ hybridization showed that expression of type II collagen gene could be observed in the cytoplasm of chondrocytic lacuna and chondrified bone tissues. mRNA in collagen of chondrocytes of the rib cartilage reached the peak level 15 days after exposure to fluoride, and decreased gradually one month and two months after exposure. Polychromatic type II collagen, breakage of collagen fiber, disorder array and reduced content of type II collagen could be found in the bone tissues with picrosirius-polarization method. CONCLUSIONS: Excessive intake of fluoride could lead to changes in types and structure of collagen (cross-linkage) of bone tissues, which caused expression of type II collagen gene in the chondrified bone tissues and enhanced its expression in the rib cartilage tissues.