Detection of genes known to be present on the mammalian Y chromosome was adapted for sexing mouse early embryos using the polymerase chain reaction (PCR) method. Sry and Zfy genes located in the sex-determining region of the Y chromosome were chosen for Y-specific target sequences, and DXNds3 sequence on the X chromosome was chosen for control. The two-step PCR method using two pairs of primers for each of the target sequences was employed for detecting the sequences. When DNAs of male and female mice were amplified with these primers, male-specific fragments were detected even in DNAs that were equivalent in amount to two cells. Mouse embryos at the two-cell stage were separated into two individual blastomeres, and one blastomere was karyotyped at the second cleavage. The remaining blastomere was subjected to PCR amplification immediately or after having been cultured for 48 h up to the morula stage. The Sry and Zfy sequences were detected in about half the embryos; detection of the Sry and Zfy sequences corresponded exactly to the presence of the Y chromosome, except in one sample of male morula in which embryos may have been lost before the PCR amplification. It is concluded that the sex of mouse preimplantation embryos can be accurately determined through detection of the Y-specific sequences using the two-step PCR method, even with the single blastomeres separated at the two-cell stage.
A new rat mutant showing severe male hypogonadism (hgn for the gene symbol) was found and isolated from the hereditary hydronephrosis rat strain originating from the Wistar-Imamichi rat. The affected rats have very tiny testes that weigh 26 mg in the adult, and contain small numbers of seminiferous tubules with degenerated Sertoli cells. It is difficult to identify the gonocyte in the seminiferous tubules in the mutant testis. Very small male accessory sex organs are all present in the mutant. The number of chromosomes is 42 (40A + XY). The structure of each chromosome is normal, showing that the mutant has male sex genes. Thus, it was considered that this status is not due to either lack of the H-Y antigen or Muellerian inhibiting factor or expression of the Tfm. By analyzing the pedigree of the matings producing the mutant, it was concluded that the status was expressed only in the male, but inherited with a single autosomal recessive trait with existence of the phenotypically normal fertile female recessive homozygote. A possible deficiency of certain known or unknown testicular growth or differentiating factor(s) in the mutant is suggested.
A new rat mutant showing aspermia was investigated. Groups of 4-7 mutant male rats were killed at 3, 5, 10, 15 and 25 weeks of age. Examination by microscope showed apparent abnormalities in the seminiferous epithelium from 3 weeks of age onward. Inclusion-like bodies were observed in the cytoplasm of pachytene spermatocytes from 3 weeks old and instead of spermiation, polynuclear giant cells were formed within the seminiferous epithelium after 5 weeks of age. Histological analysis of seminiferous epithelium of adult mutant rats also showed a marked decrease in the number of preleptotene, leptotene and pachytene spermatocytes and tubules containing only spermatogonia and Sertoli cells in the seminiferous epithelium increased with age. However, the combination of other cellular elements of germ cells in the seminiferous epithelium was similar to that in normal rats and the distribution rate of these seminiferous tubules was close to that of normal rats, indicating that cyclicity of seminiferous epithelium was still maintained in the mutant rats despite the lack of spermiation. Plasma concentrations of FSH and LH were significantly higher in the mutant male rats than in normal male rats at 5 and 10 weeks of age onward, respectively. Plasma concentrations of testosterone were lower in the mutant male rats than in normal male rats. Silastic capsules containing testosterone were implanted into the unilateral testis of adult mutant male rats and animals were autopsied 5 weeks later. However, intratesticular administration of testosterone did not affect restoration of spermatogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
A new rat line (TS inbred rats) showing uni- or bilateral ectopic scrota in about 70% of the males was established. Ectopic scrota were elongated pouches of the thin muscle layer under the suprainguinal body skin, in which hypoplastic testes and epididymides were seen. Genetic analysis revealed that the ectopic scrotum was controlled by multiple genes. Groups of 5 normal, uni- and bilaterally affected rats were killed at 25 weeks of age. Histologically, ectopic testes of uni- and bilaterally affected rats showed arrest of spermatogenesis at the primary spermatocyte stage. Contralateral testes of unilaterally affected rats were normally descended and showed normal spermatogenesis. No abnormality was seen in the urinary system. Plasma concentrations of FSH were significantly elevated in bilaterally affected rats but plasma concentrations of testosterone and LH were unchanged in uni- and bilaterally affected rats. Pituitary contents of LH and FSH were significantly elevated in bilaterally affected rats. The endocrinological status of TS inbred rats was therefore similar to that of experimentally induced cryptorchid rats.
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