Spermatozoa attain the ability to fertilize an ovum by incubation in the reproductive tract for a given period of time (capacitation). Recently Bedford & Shalkovsky (1967) and Bedford (1967) have indicated that the final stages of capacitation may be species-specific, normally occur in the oviduct and require from 0·5 to 2·5 hr in the rabbit. Capacitation enables the spermatozoon to penetrate the cumulus oophorus, corona radiata and to initiate fertilization. The work of Dukelow, Chernoff & Williams (1967) suggested that the final stages of capacitation may involve an ovum-sperm interaction occurring on the surface of, or within, the zona pellucida. These workers have suggested that the oviducal environment was detrimental to the fertility of uterine capacitated spermatozoa resident in the oviduct for several hours before ovulation. The objectives of the present study were to determine (1)
W. RICHARD DUKELOW and WILLIAM L. WILLIAMS
V. K. BHALLA, W. L. TILLMAN and W. L. WILLIAMS
The concept of sperm acrosomes as modified lysosomes which evolved to facilitate fertilization in multicellular organisms has recently been proposed (Allison & Hartree, 1968, 1970). The following lysosomal enzymes have been found in sperm acrosomes: hyaluronidase and neuraminidase (Hartree & Srivastava, 1965; Srivastava, Adams & Hartree, 1965; Srivastava, Zaneveld & Williams, 1970), β-N-acetyl glucosaminidase (Conchie & Mann, 1957), acid phosphatase, phospholipase A and aryl sulphatase (Allison & Hartree, 1970), aryl amidase (Meizel & Colham, 1972) and acrosomal proteinases (Stambaugh & Buckley, 1968, 1969; Zaneveld, Srivastava & Williams, 1969; Zaneveld, Polakoski & Williams, 1972; Polakoski, Zaneveld & Williams, 1972). This paper concerns a lysosomal enzyme which cleaves the linkage between aspartate and N-acetyl glucosamine using the synthetic substrate, 2-acetamido-l-(l-aspartamido) 1,2 dideoxy glucose (Cyclo Chemical Corp., Box 71557, Los Angeles, Calif.). The enzyme, β-aspartyl
W. RICHARD DUKELOW, H. N. CHERNOFF and W. L. WILLIAMS
Seminal plasma from eight species was assayed for decapacitation factor (df) activity. Seminal plasma from the bull, boar, stallion, rabbit and monkey all contained df activity whereas human, rooster and dog seminal plasma did not. The hypothesis is suggested that the presence, or absence, of df in the seminal plasma of a species is indicative of the need for, or lack of, capacitation within that species. Incubation of partially purified df with hyaluronidase, glucose oxidase, lysozyme or Pronase does not affect df activity ; however, such activity was destroyed by incubation with α- or β-amylase. Sialic acid, sialyl lactose and maltose were all ineffective in causing decapacitation or in preventing subsequent decapacitation of uterine sperm.
L. J. D. ZANEVELD, P. N. SRIVASTAVA and W. L. WILLIAMS
The existence of a trypsin-like enzyme (TLE) in acrosomes of epididymal spermatozoa was confirmed and was further demonstrated to be present in acrosomes of ejaculated and capacitated spermatozoa. TLE rapidly removes the zona pellucida of the ovum. Extracts of acrosomes of ejaculated spermatozoa contain an inhibitor that is separated from the TLE by purification of the TLE.
The inhibitor of TLE is also present in seminal plasma. This enzyme— enzyme inhibitor relationship appears analogous to the corona-removing enzyme-decapacitation factor relationship and part of capacitation very likely involves removal of the inhibitor from TLE and decapacitation factor from the corona-removing enzyme. TLE is inhibited by soybean trypsin inhibitor and less effectively by mercaptoethanol.
L. J. D. ZANEVELD, R. T. ROBERTSON, M. KESSLER and W. L. WILLIAMS
Treatment of capacitated rabbit spermatozoa with pancreatic trypsin inhibitor or partially purified seminal plasma trypsin inhibitor and subsequent insemination of such spermatozoa into the oviducts of ovulated rabbits markedly inhibited fertilization. Washing the spermatozoa to remove excess inhibitor did not affect the antifertility action of pancreatic trypsin inhibitor. Seminal plasma trypsin inhibitor was purified by specific binding to a trypsin-maleic anhydride-ethylene copolymer and Sephadex G-25 and G-50 column chromatography. A 500-fold purification was obtained.
Y. Zhao, L. M. Williams, L. T. Hannah, A. W. Ross, W. A. C. McKelvey and J. J. Robinson
Immunocytochemistry was used to detect the presence of oestrogen and progesterone receptors in the cervices of prepubertal lambs, seasonally anoestrous ewes, cyclic ewes, and pregnant ewes of known gestational stages, to define the roles of gonadal steroids in cervical function. The presence of the immediate early gene product, c-Fos, a marker for cellular activation, was also investigated using immunocytochemistry and in situ hybridization. Oestrogen receptor immunoreactivity was restricted to the endometrium on days 0–3 of the oestrous cycle (day 0 = oestrus). In immature animals, very few scattered nuclei in the endometrium were immunoreactive. Oestrogen receptor immunoreactivity was not apparent in the endometrium during the remainder of the oestrous cycle or in this region in anoestrous animals. In pregnant ewes, oestrogen receptor immunostaining appeared as relatively few isolated nuclei in the connective tissue stroma. Progesterone receptor immunoreactivity was found in the endometrium at days 0–3 of the oestrous cycle and also in the luminal epithelium, the myometrium and the blood vessels. Progesterone receptor immunoreactivity was also found in these regions, with the exception of the endometrium, at all other stages examined. Immunostaining for c-Fos was present in the endometrium at days 0–3 of the oestrous cycle, and some scattered immunopositive nuclei were present in prepubertal animals. c-Fos immunoreactivity was also found in the myometrium and in blood vessels at all other stages examined. Visualization of c-fos gene expression by in situ hybridization showed that it occurred in the luminal epithelium and blood vessels at oestrus, but was restricted to the blood vessels in all other samples examined.
M Amstalden, D A Zieba, M R Garcia, R L Stanko, T H Welsh Jr, W H Hansel and G L Williams
Experiments were performed to test the hypothesis that lamprey GnRH-III (lGnRH-III) selectively releases FSH. Primary cultures of bovine adenohypophyseal cells were treated with mammalian GnRH (mGnRH) and lGnRH-III (10−9, 10−8, 10−7 and 10−6 M) or control media in Experiment 1. All doses of mGnRH and the two highest doses of lGnRH-III stimulated (P < 0.001) a non-selective release of LH and FSH. In Experiments 2–4, Latin Square designs were utilized in vivo to examine whether physiological and hormonal milieu regulate putative selective effects of lGnRH-III. In Experiments 2 and 3, ovariectomized cows with basal levels of estradiol only (Experiment 2) or in combination with luteal phase levels of progester-one (Experiment 3) were injected with mGnRH and lGnRH-III (0.055, 0.11, 0.165 and 1.1 μg/kg body weight (BW) and saline. All doses of mGnRH released (P < 0.001) LH and FSH, but only the highest dose of lGnRH-III stimulated (P < 0.001) a non-selective release of both LH and FSH (Experiment 3). For Experiments 4A and 4B, intact, mid-luteal phase cows were injected with mGnRH and lGnRH-III (1.1 μg/kg BW; Experiment 4A), lGnRH-III (1.1 and 4.4 μg/kg BW; Experiment 4B) and saline. As before, mGnRH released (P < 0.001) both LH and FSH at all doses. In contrast, lGnRH-III at the highest dose released (P < 0.001) LH but not FSH. These findings suggest that lGnRH-III may act as a weak competitor for the mGnRH receptor and do not support the hypothesis that it selectively releases FSH in cattle.