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The effects of zona pellucida glycoproteins, sulfated polymers and non-sulfated polymers on the activation kinetics of boar sperm proacrosin to β-acrosin have been investigated. The aim has been to understand more about the behaviour and function of this protein after it has been released from the acrosome at the time of fertilization. Purified proacrosin was allowed to autoactivate at pH 8.0 in the presence of different concentrations of homologous zona glycoproteins, sulfated polymers (fucoidan, chondroitin sulfates A, B and C, dextran sulfate, polyvinylsulfate and heparin) and non-sulfated polymers (dextran, polyvinylphosphate and hyaluronic acid). Enzyme activity was measured against N-benzoyl-l-arginine p-nitroanalide substrate and changes in molecular mass of the protein monitored by SDS-PAGE. Results show that zona pellucida glycoproteins, fucoidan, dextran sulfate and polyvinylsulfate all potentiate the conversion of proacrosin to β-acrosin but subsequently inhibit its amidase activity. Dextran, polyvinylphosphate, chondroitin sulfates A, B and C and glucose-6-sulfate, on the other hand, either have no effect on autoactivation and β-acrosin activity, or enhance it slightly. SDS-PAGE analysis confirmed these observations and further indicated that binding of sulfated polymers to proacrosin inhibited staining by Coomassie Blue. These results are consistent with the hypothesis that binding of zona pellucida glycoproteins and sulfated compounds to proacrosin/acrosin is stereospecific and that contact activation onto soluble 'surfaces' causes conformational changes that are responsible for potentiation or inhibition of activation. The implications of these findings for sperm binding and penetration of the zona pellucida are discussed.
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Search for other papers by A. H. WILLIAMS in
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Intermittent teasing of ewes with a ram has been reported both to increase (Zeltobrjuh & Rak, 1965) and to decrease (Parsons, Hunter & Rayner, 1967) the interval from the onset of oestrus to ovulation.
Parsons et al. (1967) suggested that the presence of the ram influenced the time of ovulation in the ewe by altering the rate of the secretion of the LHreleasing factor. Cumming, Buckmaster, Blockey, Goding, Winfield & Baxter (1971, 1973) investigated the relationship between the preovulatory release of LH and ovulation and found that ewes ovulated between 21 and 26 hr (mean 23·5 hr) after the release of LH. Blockey (1971), however, found this interval to vary from 23 to 55 hr, the mean being 30 hr. While Cumming et al. (1973) removed the ewes from contact with the ram during oestrus, Blockey (1971) used intermittent teasing.
It is possible that association of the sexes during oestrus
Department of Animal Sciences, University of Florida, Gainesville, Florida, USA
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Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, Florida, USA
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Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, Florida, USA
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Department of Animal Sciences, University of Florida, Gainesville, Florida, USA
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The microenvironment of the ovarian follicle is key to the developmental success of the oocyte. Minor changes within the follicular microenvironment can significantly disrupt oocyte development, compromising the formation of competent embryos and reducing fertility. Previously described as a sterile environment, the ovarian follicle of women has been shown to contain colonizing bacterial strains, whereas in domestic species, pathogen-associated molecules are concentrated in the follicular fluid of animals with uterine infection. The aim of this study is to determine whether human granulosa–luteal cells mount an innate immune response to pathogen-associated molecules, potentially disrupting the microenvironment of the ovarian follicle. Human granulosa–luteal cells were collected from patients undergoing assisted reproduction. Cells were cultured in the presence of pathogen-associated molecules (LPS, FSL-1 and Pam3CSK4) for 24h. Supernatants and total RNA were collected for assessment by PCR and ELISA. Granulosa–luteal cells were shown to express the molecular machinery required to respond to a range of pathogen-associated molecules. Expression of TLR4 varied up to 15-fold between individual patients. Granulosa–luteal cells increased the expression of the inflammatory mediators IL1B, IL6 and CXCL8 in the presence of the TLR4 agonist E. coli LPS. Similarly, the TLR2/6 ligand, FSL-1, increased the expression of IL6 and CXCL8. Although no detectable changes in CYP19A1 or STAR expression were observed in granulosa–luteal cells following challenge, a significant reduction in progesterone secretion was measured after treatment with FSL-1. These findings demonstrate the ability of human granulosa–luteal cells to respond to pathogen-associated molecules and generate an innate immune response.
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Search for other papers by W. L. WILLIAMS in
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Summary.
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.
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Summary.
The cortical granules of hamster ova were ruptured in vitro by electrical stimulation (standard treatment was 150 V, 1·0 msec). Approximately 8 min were required for the cortical granule material so released to induce a complete zona reaction. The active material from the cortical granules was released into the medium from vitelli and was shown to act directly on the zona pellucida and on mouse, as well as on hamster, ova, i.e. its action is not species specific. The action of the cortical granule material was reversed by trypsin inhibitors. Such a tryspin-like protease, causing the zona reaction, was also released from vitelli by spermatozoa.
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At birth, the physiological role of prostaglandins in bitches is unclear. Bitches were treated before parturition with either saline, the prostaglandin analogue, sodium cloprostenol, or the prostaglandin synthetase inhibitor, flunixin meglumine. The animals were examined regularly to determine the onset of parturition and a series of blood samples were taken to define the hormonal profiles before, during and after birth. Animals treated with cloprostenol whelped earlier than did controls. In addition, the prostaglandin F2α metabolite surge and decrease in plasma progesterone concentration and rectal temperature were earlier than in controls. Flunixin meglumine disrupted the normal 13,14-dihydro-15-keto prostaglandin F2α profile but did not abolish prostaglandin synthesis completely or delay the onset of labour in treated animals. This study confirms that prostaglandins induce luteolysis and the onset of labour in the bitch. However, the partial inhibition of prostaglandin synthesis does not prevent parturition.
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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.
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Two experiments were conducted in cyclic beef heifers to determine whether active immunization against bovine inhibin α 1–26 Gly-Tyr (bINH) affected follicular dynamics, hormone concentration or ovulation rate. In Expt 1, heifers (n = 9) were actively immunized against bINH conjugated to human α globulins (HAG) using bisdiazotized benzidine in non-ulcerative Freund's adjuvant (NUFA; primary on day 0; booster injections on days 53, 84 and 116 using conjugated bINH and on days 176 and 366 using unconjugated bINH; ten heifers were used as controls). Ovaries were examined daily using ultrasound scanning (days 70–155 and 384–391) and corresponding blood samples were collected for bINH antibody titre, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestradiol determinations. Four treated and four control heifers were injected with 10 μg gonadotrophin-releasing hormone (GnRH) on day 386 (day 2 of the oestrous cycle). Although bINH-immunized heifers had variable antibody titres ranging from 4 to 50% I125-labelled bINH bound to serum diluted 1:2000, ovulation rate was unaffected. In oestrous cycles with three dominant follicles, the ovulatory follicles grew faster (2.5 ± 0.2 versus 1.6 ± 0.3 mm day−1; mean ± sem), had shorter durations of growth (5.7 ± 0.8 versus 9.6 ± 1.6 days) and duration of detection (7.5 ± 0.8 versus 12.0 ± 2.4 days) in immunized heifers. Mean concentrations of FSH, LH and oestradiol were unaltered in most cases during oestrous cycles in bINH-immunized compared with control heifers. There was no significant difference in the percentage increase in FSH or LH, after GnRH injection, between control and immunized heifers. As ovulation rate was unaltered in the first experiment, a second similar study was designed using a different immunization protocol. In Expt 2, heifers were immunized with bINH conjugated to human serum albumin using glutaraldehyde with the following doses: 0.0 (control; n = 7), 0.33 (n = 7), 1.0 (n = 8) and 3.0 (n = 7) mg. Three booster immunizations were given 33, 66 and 209 days after primary immunization. Immunization increased the number of oestrous cycles with multiple ovulations (42 of 132 (32%) oestrous cycles examined) compared with controls (1 of 30 (3.3%) oestrous cycles examined). Neither titre nor ovulation rate was affected by dose of bINH used. In summary, following bINH immunization, ovulation rate was not increased despite changes in follicular dynamics in Expt 1, but was increased in 32% of oestrous cycles in Expt 2. We conclude that immunization protocols can affect responsiveness of heifers to bINH immunization, and that immunization against inhibin can increase ovulation rate.
