Intercellular pathway of leucine catabolism in rat spermatogenic epithelium.

A unique intercellular pathway of leucine catabolism was observed in vitro in rat spermatogenic epithelium. Sertoli cells convert leucine via transmination into 4-methyl-2-oxovalerate, and spermatocytes and spermatids reduce exogenous 4-methyl-2-oxovalerate to 2-hydroxy-4-methylvalerate, which is then released by the spermatogenic cells. The NADH-dependent reduction of 4-methyl-2-oxovalerate could be catalysed by the male-germ-cell-specific lactate dehydrogenase isoenzyme LDH-C4 in the cytosol of the spermatogenic cells, concomitant with the NAD+-dependent conversion of exogenous lactate into pyruvate.     

Expression of the cDNA encoding for mouse sperm-specific lactate dehydrogenase C in Chinese-hamster ovary cells.

The cloned cDNA encoding for mouse sperm-specific lactate dehydrogenase C (LDH-C) was inserted immediately downstream to the MMTV 5' LTR promoter, and it was shown to synthesize mouse LDH-C polypeptide in Chinese-hamster ovary cells. The mouse LDH-C subunit and the endogenous Chinese-hamster LDH-A subunit formed in vivo a heterotetrameric LDH-A3C1 isoenzyme, and this novel isoenzyme exhibited enzymic activity utilizing lactate as substrate.     

Differential effects of (+)- and (-)-gossypol enantiomers on LDH-C4 activity of hamster spermatogenic epithelium in vitro

Tubular fragments (spermatogenic epithelium) from immature hamsters were isolated and cultured with low doses of (+)- and (-)-gossypol enantiomers. The activity of lactate dehydrogenase isoenzyme LDH-C4 was estimated as a marker for spermatogenic cell development and alpha-ketoisovalerate was used as the substrate. In the absence of gossypol, the specific activity of LDH-C4 in the tubular fragments was increased 40% during incubation for 48 h. This developmental increase was suppressed by gossypol. The specific activity of LDH-C4 in the tubular fragments was lowered by gossypol, after 48 h of culture in the presence of low doses of racemic gossypol (1-4 microM) and 1% fetal calf serum. In this in-vitro system the (-)-enantiomer but not the (+)-enantiomer of gossypol affected the LDH-C4 activity. This is in agreement with other reports showing that only the (-)-enantiomer induces infertility. The observed action of gossypol on LDH-C4 activity in the tubular fragments may reflect gossypol-induced degeneration of spermatogenic cells. The present in-vitro system can be used to estimate the actions of gossypol derivatives, other investigational antifertility agents, and toxic agents on the spermatogenic epithelium.     

Acrosin biosynthesis in meiotic and postmeiotic spermatogenic cells.

It has been widely accepted that mammalian sperm acrosin is first synthesized only in the postmeiotic stages of spermatogenic cells. In this study, we carried out Northern blot analysis of RNAs prepared from purified populations of mouse spermatogenic cells. The acrosin mRNA was obviously found in meiotic pachytene spermatocytes, and the mRNA content markedly increased in postmeiotic round spermatids. Also, the acrosin mRNA in pachytene spermatocytes was functionally associated with polysomes. These results provide evidence that acrosin biosynthesis is already started in meiotic cells and continues through the early stages of spermiogenesis.     

Quantification of somatic and spermatogenic cell proliferation in the testes of ruminants, using a proliferation marker and flow cytometry analysis

We compared 2 methods for the quantification of proliferation in somatic and spermatogenic compartments of post mortem-collected testes in cattle and roe deer. Proliferation was evaluated by estimation of the tissue polypeptid specific antigen (TPS) using an ELISA. This proliferation-specific marker was detected in homogenized cells after selective enrichment of different cell types by density gradient centrifugation. The haploid, diploid and tetraploid cells were monitored by one-parameter flow cytometry and analyzed for mitotic cell cycle. Somatic and spermatogenic cells were discriminated by dual-parameter flow cytometry after DNA staining with propidium iodide and selective labelling of stromatic cells with a vimentin antibody. The TPS was related to the ploidy of cells and their somatic or spermatogenic type. High concentrations of TPS were found in both species. The TPS values varied with different contents of spermatogenic and somatic cells in the fractions of the density gradient. The TPS was positively correlated with spermatogenic cells in the G2/M phase of mitotic cycle (r = 0.474; P < 0.01) and negatively correlated with somatic cells (r = -0.676; P < 0.0001) in roe deer (n = 40). Discrimination of germinative and stromatic cells in the G2-M phase showed their varying proliferation during the annual cycle in roe deer. The quantification of tetraploid spermatogenic cells allowed the calculation of an exact meiotic transformation (ratio haploid:tetraploid cells). In conclusion, TPS indicates proliferation in the germinative compartment of the testes. However, this marker provides only relative values, without information on the number and type of proliferating cells. Dual-parameter flow cytometry using specific staining for vimentin proves to be a better method for studying changing mitotic and meiotic steps during the involution and recrudescence of testes in seasonally breeding ruminants, as it relates proliferative processes directly to both spermatogenic and somatic cells.     

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.