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research-article |
Institutes of Reproductive Medicine4 and Biology,5 University of Münster, D-48129 Münster, Germany
ABSTRACT
Spermatozoa need to undergo regulatory volume decrease (RVD) upon ejaculation to counteract swelling due to the hypo-osmolality of female tract fluids. Defects in sperm RVD lead to failure in both cervical mucus penetration in humans and utero-tubal junction passage in mice. The role of K/Cl cotransporters (KCCs) in RVD was investigated by incubation of spermatozoa from the murine cauda epididymidis and from human ejaculates in media mimicking female tract fluid osmolalities in the presence of KCC inhibitors. Furosemide at 100 µM or more caused swelling of murine spermatozoa as detected with a flow cytometer by increased laser forward scatter over 30 to 75 min of incubation. Bumetanide, known to have low affinity for KCCs, was effective at 1 mM, whereas 10 µM and 20 µM of the specific inhibitor DIOA (dihydroindenyl-oxy alkanoic acid) increased cell volume. These drug doses were ineffective in human spermatozoa, which, however, responded to quinine, confirming the occurrence of RVD under control conditions. The molecular identity of the murine KCC isoform involved was determined at both mRNA and protein levels. Conventional RT-PCR indicated the presence of transcripts from Slc12a4 (KCC1), Slc12a6 (KCC3), and Slc12a7 (KCC4) in the testis, whereas RT-nested PCR revealed the latter two isoforms in sperm mRNA. Of these three isoforms, only SLC12A7 (KCC4) was detected in murine sperm protein by Western blotting. Therefore, besides organic osmolyte efflux and KCl release through separate K+ and Cl– ion channels, SLC12A7 also is involved in murine but not human sperm RVD mechanisms.
fertilization, gamete biology, sperm, sperm motility and transport
3Current address: ZMNH Centre for Molecular Neurobiology, Institute for Neural Signal Transduction, Hamburg, Germany.
1Supported by the Deutsche Forschungsgemeinschaft (grant no. YE37/6-1.
Correspondence: 2 C.H. Yeung, Institute of Reproductive Medicine, Domagkstrasse 11, D-48129 Münster, Germany. FAX: 49 251 835 6093; e-mail: chinghei.yeung{at}ukmuenster.de
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