The lactate dehydrogenase (LDH) protein family members characteristically are distributed in tissue- and cell type-specific patterns and serve as the terminal enzyme of glycolysis, catalyzing reversible oxidation reduction between pyruvate and lactate. They are present as tetramers, and one family member, LDHC, is abundant in spermatocytes, spermatids, and sperm, but also is found in modest amounts in oocytes. We disrupted the Ldhc gene to determine whether LDHC is required for spermatogenesis, oogenesis, and/or sperm and egg function. The targeted disruption of Ldhc severely impaired fertility in male Ldhc(-/-) mice but not in female Ldhc(-/-) mice. Testis and sperm morphology and sperm production appeared to be normal. However, total LDH enzymatic activity was considerably lower in Ldhc(-/-) sperm than in wild type sperm, indicating that the LDHC homotetramer (LDH-C(4)) is responsible for most of the LDH activity in sperm. Although initially motile when isolated, there was a more rapid reduction in the level of ATP and in motility in Ldhc(-)(/-) sperm than in wild-type sperm. Moreover, Ldhc(-/-) sperm did not acquire hyperactivated motility, were unable to penetrate the zona pellucida in vitro, and failed to undergo the phosphorylation events characteristic of capacitation. These studies showed that LDHC plays an essential role in maintenance of the processes of glycolysis and ATP production in the flagellum that are required for male fertility and sperm function.

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture system to maintain viable spermatogonia for long periods of time. Here we report the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem cell factor. This differentiation can occur in the absence of supportive cells. The immortalized spermatogonial cell line may serve as a powerful tool in elucidating the molecular mechanisms of spermatogenesis. Furthermore, through genomic modification and transplantation techniques, this male germ cell line may be used to generate transgenic mice and to develop germ cell gene therapy.

E sablhing mma germ-cell lines capable ofdifferentiation in vgo would greatiy facilitate the study of gmetogene and the meiotic process that is so fundamental for reproduction and the maintenance ofgenetic diversity ofthe species. We have established two germ-cell lie and MATERIALS AND METHODSCell Lines. A single cell suspension was obtained from decapsulated testes of 6-week-old BALB/c mice and prepared as described (7). A cell fraction enriched with preleptotene spermatocytes was then isolated by the STA-PUT procedure and unit-gravity sedimentation at 40C (10). Ten to twelve million cells were seeded in a 60-mm tissue culture dish (Falcon) with S ml of complete CMRL-1066 medium (7) and immediately transfected. Cotransfection of 12.5 pig of pSV3neo (11) (American Type Culture Collection, no. 37150) and 12.5 ug ofLTRpS3cG9 (12) (kindly provided by Channing Der, University of North Carolina, Chapel Hill) per dish was performed by the calcium phosphate method (13). Cells were cultivated for 7 days at 37C and 5% CO2 before selection. G418 (Geneticin; GIBCO) at an active concentration of 200 jg/ml was used for the selection of neomycin-resistant colonies. Genomic DNA and total RNA were isolated from established cell lines and mouse testis by standard methods (14). Soft-agar cultures were established by the method of Hamburger and Salmon (15) as described (7).Immunocytochemistry. and GC-3spc(ts) cells were grown at 390C, 37C, and 320C to monolayers on LabTek slide chambers (Nunc) in Dulbecco's modified Eagle's medium supplemented as described (7) and were fixed with cold methanol, washed in phosphate-buffered saline (PBS), and incubated in PBS at 40C for at least 2 days. Cells were stained with a polyclonal antibody against the germ cell-specific isozyme of lactate dehydrogenase, C4 (16), and a polyclonal antibody against the germ cell-specific isoform of the cytochrome c, cytochrome ct (17). Both antibodies were adsorbed with the somatic form of each enzyme. Cells were also stained with a monoclonal antibody against mouse wild-type p53 (Ab-1, clone 421; Oncogene Science) or with a §To whom reprint requests should be addressed. 5533The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.