Modulation of Mammalian Sperm Activity by Reproductive-derived Spink Using Mice as Experimental Animals
Date Issued
2010
Date
2010
Author(s)
Ou, Chung-Mao
Abstract
Mice were used to study the involvement of reproductive-derived Spink from males in mammalian reproduction. A Kazal-type protease inhibitor purified from mouse seminal vesicle secretion by our group has an inhibitory constant (Ki) of 0.15 nM to trypsin and a primary structure consisting 57 amino acid residues. Since this rather small protein was derived from the P12 cDNA cloned from the mouse ventral prostate by Mills et al., it was tentatively named P12. According to the Mouse Genome Informatics nomenclature committee, P12 is now renamed mouse Spink3. Our previous study suggest that Spink3 has a single-type binding site (1.49 x 106 sites/cell) with a Kd value of 70 nM mainly on the plasma membrane overlaying the acrosomal region of mouse sperm cell. Yet, the membrane-anchored molecule of Spink3 on sperm head has not been established.
We identified a testis-specific protease-like protein tentatively named TESPL from the clones of a yeast two-hybrid screen against a mouse testicular cDNA library using the trypsin inhibitor Spink3 from male accessory sexual glands as bait. We found that TESPL transcription was restricted to the testis and that the level of transcription was positively correlated with animal maturation. Alignment of the cDNA-deduced sequences of serine proteases showed the replacement of an essential serine residue in the catalytic triad of serine proteases by a proline residue in TESPL, which was demonstrated to be a membrane-bound protein devoid of proteolytic activity. The immunohistochemical staining patterns of seminiferous tubules in the testis revealed TESPL mainly on postmeiotic cells such as spermatids and spermatozoa. On the mouse sperm from caudal epididymis, TESPL was localized mainly on the plasma membrane overlaying the acrosomal region.
Result of indirect immunofluorescence stain indicated that Spink3 was found in the secretion and seen on a considerable portion of sperm in the uterine cavity but disappeared in the oviduct lumen after coitus. The Spink3-sperm binding did not change the cell status and inhibit the capacitation-related protein tyrosine phosphorylation and cell motility enhancement, but reduced the head [Ca2+]i and the ionophore A23187-induced acrosome reaction. The sperm-egg interaction and fertility rate greatly suppressed after insemination of oocyte-cumulus complexes containing Spink3 in the capacitated sperm preparation. It is of interest to note that R19L, like its wild type, can bind sperm to suppress AR and reduce fertility. This substantiates that the reactive R19 on the Spink3 molecule for protease inhibition is not essential for its action on sperm.
Not the membrane modification associated with the sperm capacitation but the Spink3-inhibiting trypsin-like activity (SITA) in the uterine fluid of estrous females was involved in releasing Spink3 from sperm to resume their fertilizing ability. Meanwhile, suppressing SITA by free Spink3 protected sperm from proteolytic damage in the uterine cavity, manifesting the important interplay of Spink and SITA during natural coitus.
Using mice as experimental animals, this work was conducted to prove that: i) Spink3 binding on the apical hook of sperm head prevents them from becoming infertile before encountering an egg by diminishing the acrosome reaction of capacitated sperm; ii) Spink3 on sperm reduces in vitro fertility, and the Spink3-inhibiting trypsin-like activity (SITA) secreted from the uterus of estrous females during natural coitus releases Spink3 from sperm to restore their ability to fertilize; iii) the proteolytic damage to sperm from SITA is suppressed by free Spink3 in the uterine cavity.
Subjects
sperm
protease
acrosome
fertilization
capacitation
Type
thesis
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