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Transaldolase Is Essential for Maintenance of the Mitochondrial Transmembrane Potential and Fertility of Spermatozoa

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Transaldolase Is Essential for Maintenance of the Mitochondrial Transmembrane Potential and Fertility of Spermatozoa Transaldolase is essential for maintenance of the mitochondrial transmembrane potential and fertility of spermatozoa Andras Perl*†‡, Yueming Qian*, Kazim R. Chohan§, Cynthia R. Shirley¶, Wendy Amidon*, Sanjay Banerjee*, Frank A. Middletonʈ, Karina L. Conkrite*, Maureen Barcza§, Nick Gonchoroff§, Susan S. Suarez**, and Katalin Banki§ Departments of *Medicine, †Microbiology and Immunology, §Pathology, and ʈNeuroscience and Physiology, College of Medicine, State University of New York, Syracuse, NY 13210; ¶Murine Cryopreservation Laboratory, University of California, Davis, CA 95616; and **Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 Edited by Ryuzo Yanagimachi, University of Hawaii, Honolulu, HI, and approved July 27, 2006 (received for review April 2, 2006) Fertility of spermatozoa depends on maintenance of the mitochon- phase can be formulated without this enzyme (10); therefore, the drial transmembrane potential (⌬␺m), which is generated by the overall reason for existence of TAL had not been established. electron-transport chain and regulated by an oxidation–reduction To investigate the role of TAL in the PPP and mammalian equilibrium of reactive oxygen intermediates, pyridine nucleo- development in general, the genomic locus was inactivated in the Ϫ Ϫ tides, and glutathione (GSH). Here, we report that male mice mouse. Here, we report that TAL / mice develop normally; lacking transaldolase (TAL)؊/؊ are sterile because of defective however, males are sterile because of functional and structural ؊/؊ forward motility. TAL spermatozoa show loss of ⌬␺m and defects of mitochondria. mitochondrial membrane integrity because of diminished NADPH, NADH, and GSH. Mitochondria constitute major Ca2؉ stores; thus, Results diminished mitochondrial mass accounts for reduced Ca2؉ fluxing, Inactivation of the Mouse TAL (TAL-M) Gene Locus by Homologous defective forward motility, and infertility. Reduced forward pro- Recombination. We replaced a 1,116-bp EcoRI-PstI fragment har- gression of TAL-deficient spermatozoa is associated with dimin- boring adjacent parts of exon 4 and intron 4 of the TAL-M locus ished mitochondrial reactive oxygen intermediate production and with the neo gene (Fig. 7A, which is published as supporting Ca2؉ levels, intracellular acidosis, and compensatory down-regu- information on the PNAS web site). This manipulation deleted lation of carbonic anhydrase IV and overexpression of CD38 and amino acid residues 138–153, including lysine 142, which is essential ␥-glutamyl transferase. Microarray analyses of gene expression in for catalytic activity of TAL (11). The 129SvJ-derived TC1 ES cells the testis, caput, and cauda epididymidis of TAL؉/؉, TAL؉/؊, and heterozygous for the disrupted allele were microinjected into ͞ TAL؊/؊ littermates confirmed a dominant impact of TAL deficiency C57BL 6 blastocysts to generate chimeras. Chimeric males were ͞ on late stages of sperm-cell development, affecting the electron- mated with C57BL 6 females, and tail DNA of the offspring was transport chain and GSH metabolism. Stimulation of de novo GSH tested for transmission of the targeted allele by PCR (Fig. 7B), synthesis by oral N-acetyl-cysteine normalized the low fertility rate Southern blot (Fig. 7C), and Western blot analyses (Fig. 7D). of TAL؉/؊ males without affecting the sterility of TAL؊/؊ males. Among 692 pups born to heterozygote parents, the proportion of ؊ ؊ ؉ ؉ WT (168 of 692, 24.4%), heterozygote (355 of 692, 51.2%), and Whereas TAL / sperm failed to fertilize TAL / oocytes in vitro, homozygote knockout genotypes (169 of 692, 24.4%) followed the sterility of TAL؊/؊ sperm was circumvented by intracytoplasmic expected Mendelian inheritance. sperm injection, indicating that TAL deficiency influenced the structure and function of mitochondria without compromising the Complete Male Infertility in Homozygous and Partial Male Infertility nucleus and DNA integrity. Collectively, these data reveal an in Heterozygous Knockout Mice. Breeding pairs were set up between CELL BIOLOGY essential role of TAL in sperm-cell mitochondrial function and, thus, adult (Ͼ8 weeks of age) male and female mice born to different male fertility. heterozygote parents. TALϩ/Ϫ males had reduced fertility, and TALϪ/Ϫ males were completely infertile (Fig. 1). Although vaginal orward motility and fertility of spermatozoa depend on pro- plugs were observed, 15 TALϪ/Ϫ males paired with WT females Fduction of reactive oxygen intermediates (ROIs) (1) and main- produced no pregnancy over 5 months. TALϪ/Ϫ males displayed no tenance of the mitochondrial transmembrane potential (⌬␺m)(2, obvious differences in weight and development. Weight and light- 3). ⌬␺m is generated by the electron-transport chain and subject to microscopic morphology of their reproductive organs (testes, sem- regulation by an oxidation–reduction equilibrium of ROI, pyridine nucleotides (NADH͞NAD ϩ NADPH͞NADP), and reduced glu- tathione (GSH) (4). In turn, NADPH, a reducing equivalent Author contributions: A.P., Y.Q., K.R.C., C.R.S., and K.B. designed research; A.P., Y.Q., W.A., K.R.C., C.R.S., S.B., F.A.M., K.L.C., M.B., N.G., S.S.S., and K.B. performed research; A.P., Y.Q., required for biosynthetic reactions and regeneration of GSH from W.A., K.R.C., C.R.S., S.B., F.A.M., S.S.S., and K.B. analyzed data; and A.P. and K.B. wrote the its oxidized form, is produced by the pentose phosphate pathway paper. (PPP) (5). The PPP was originally formulated based on metabolites The authors declare no conflict of interest. and enzymes detected in yeast (6). Thus, PPP comprises two This paper was submitted directly (Track II) to the PNAS office. separate oxidative and nonoxidative phases. Enzymes of the oxi- Abbreviations: CAIV, carbonic anhydrase IV; ⌬␺m, mitochondrial transmembrane potential; dative phase, glucose 6-phosphate dehydrogenase (G6PD) and G6PD, glucose 6-phosphate dehydrogenase; GGT, ␥-glutamyl transpeptidase; GSH, gluta- 6-phosphogluconate dehydrogenase, can generate both ribose thione; ICSI, intracytoplasmic sperm injection; NAC, N-acetyl cysteine; PPP, pentose phos- 5-phosphate (R5P) and NADPH. Although enzymes of the non- phate pathway; ROI, reactive oxygen intermediate; S7P, sedoheptulose 7-phosphate; TAL, transaldolase; TK, transketolase. oxidative phase, transketolase (TK) and transaldolase (TAL), can Data deposition: The sequence reported in the paper has been deposited in the GenBank convert R5P into glucose 6-phosphate (G6P) for the oxidative database (accession no. AY466103). phase, and, thus, indirectly, these enzymes can also contribute to the ‡To whom correspondence should be addressed at: Department of Medicine, State Uni- generation of NADPH, the significance of the nonoxidative branch versity of New York, 750 East Adams Street, Syracuse, New York 13210. E-mail: perla@ is less well established. Certain organisms (7, 8) and mammalian upstate.edu. tissues do not express TAL (9), and, significantly, the nonoxidative © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0602678103 PNAS ͉ October 3, 2006 ͉ vol. 103 ͉ no. 40 ͉ 14813–14818 Downloaded by guest on September 27, 2021 Fig. 1. Male infertility in TAL-deficient mice. Fifteen male and 15 female mice of each genotype were paired, with a single WT female or male per cage. Values indicate mean number Ϯ SD of pups per breeding pair recorded for four generations. Based on comparison with TALϩ/ϩ controls, P values Ͻ0.05 are shown. inal vesicles, prostate, and epididymis) did not differ from those of spectra of potentiometric-, oxidation-, or Ca2ϩ-sensitive fluorescent TALϩ/Ϫ and TALϩ/ϩ littermates. Sperm of all developmental probes (15, 16). Although overall viability of TALϪ/Ϫ,TALϩ/Ϫ,or ϩ/ϩ Ϫ/Ϫ stages were present in similar numbers in the testes and efferent TAL spermatozoa was not different, ⌬␺m of TAL sperma- ducts. Complete or partial TAL deficiency did not influence female tozoa was profoundly diminished (Fig. 2 and Fig. 10, which is fertility (Fig. 1). published as supporting information on the PNAS web site). ⌬␺m of TALϩ/Ϫ spermatozoa was reduced to a lesser extent. Mitochon- Diminished Forward Motility and in Vitro Fertility of TAL-Deficient drial mass in TALϪ/Ϫ spermatozoa was diminished, as determined Spermatozoa. The motility of spermatozoa was investigated by using by NAO and MTG fluorescence. The extent of ⌬␺m loss, as a computer-assisted semen-analysis system. Although no significant determined by DiOC6 fluorescence, exceeded the loss of mitochon- differences were observed in total numbers and viability of sper- drial mass detected by NAO and MTG fluorescence. Reduced JC-1 matozoa, motility parameters, including progressive velocity FL-2͞FL-1 fluorescence also suggested loss of ⌬␺m independent of (straight line velocity) and track speed were markedly reduced, mitochondrial mass. Ϫ/Ϫ whereas beat frequency of TAL sperm was increased (Table 1). Maintenance of ⌬␺m is required for generation of ROI (17); thus, Tracings of video images revealed stiffened flagellar midpieces of reduced ⌬␺m may lead to diminished ROI production by TAL- TALϪ/Ϫ sperm (Fig. 8, which is published as supporting informa- deficient sperm cells. As shown in Fig. 2, ROI levels, monitored by tion on the PNAS web site). We attempted to rescue infertility of dihydroethidium (hydroethidine, DHE) as well as by dihydrorho- TALϪ/Ϫ sperm by in vitro fertilization. Sperm of four TALϪ/Ϫ mice damine and dichlorofluorescein (data not shown) (15), were di- failed to fertilize
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