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Ministry of Agriculture, Fisheries and Food, Cattle Breeding Centre, Shinfield, Reading RG2 9BZ
(Received 4th November 1974)
One of the earliest visual signs of sperm damage is the loss of acrosomal integrity. Consequently, assay of the release into the seminal plasma of hyaluronidase, which is localized in the acrosome (Mancini, Alonsa, Bacquet, Avarez & Nemirosky, 1964; Gould & Bernstein, 1973) should provide an early and sensitive indication of damage sustained by the spermatozoa during processing of semen for artificial insemination (A.I.). Previous biochemical tests of sperm viability have included the assay of phenylalanine α-ketoglutarate transaminase released into boar seminal plasma (Gemert, Hendrike & Van der Horst, 1972). A correlation was found between high enzyme activity in seminal plasma and poor fertility. The release of glutamic oxaloacetic transaminase has been considered an indication of cell damage (Brown, Crabo, Graham & Pace, 1971) and assay of this non-acrosomal enzyme has been investigated
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The guinea-pig has an oestrous cycle of 16 to 18 days. Many original observations in reproductive endocrinology such as formation of deciduoma in response to trauma (Loeb, 1907), existence of the oestrous cycle (Stockard & Papanicolaou, 1917) and the ability of the uterus to modify luteal function (Loeb, 1923) involved the guinea-pig as the experimental animal. Several studies have delineated the pattern of progesterone secretion during the oestrous cycle of guinea-pigs (Heap, Perry & Rowlands, 1967; Feder, Resko & Goy, 1968; Challis, Heap & Illingworth, 1971; Blatchley, Donovan, Horton & Poyser, 1972) but comparative changes in the secretion of oestrogens have received very little attention. Challis et al. (1971) attempted to measure total oestrogens by radioimmunoassay in the peripheral plasma, but failed to detect any due to their very low levels. In the present study, we determined
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Summary.
The use of the pH-pill has allowed continuous monitoring of vaginal pH during human coitus. In the case of a couple of normal fertility, there was an immediate buffering by seminal plasma so that the vaginal pH changed in 8 sec from pH 4·3 to pH 7·2. In the case of a couple of low (male) fertility, the immediate effect of the arrival of semen in the vagina was a change from pH 3·5 to pH 5·5. A similarly small change in pH occurred when the seminal volume of a fertile male subject had been depleted, by repeated ejaculation, to 1 ·5 ml. At this pH (5·5), spermatozoa are generally immobilized.
It has been possible to alter normal fertility, as judged by postcoital tests for sperm motility, by the introduction of a pH 3·6 buffer solution into the vagina before coitus. In this latter experiment, the vaginal pH immediately after ejaculation was 5·0 and, after 2 hr, had reached pH 5·4.
These results in vivo suggest that the vagina is not the hostile environment hitherto imagined, since the normal ejaculate readily overcomes the vaginal acidity by its powerful buffering action. Low seminal volume, with or without a concomitantly low sperm count, and artificial changes in vaginal environment by buffer solutions may affect fertility.
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After mating, mammalian spermatozoa are transported to the lower oviductal isthmus. Spermatozoa are sequestered at the isthmus by attaching and interacting with oviductal epithelial cells, hence forming a sperm reservoir. In several mammalian species, specific carbohydrates mediate sperm-oviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig sperm-oviductal epithelial cell interactions. This assay was validated. The sensitivity of the assay was such that it was possible to discriminate between different sperm concentrations and sperm-oviductal epithelial cell co-incubation periods, spermatozoa with damaged plasma membranes and epithelial cells of non-reproductive origin. Optimal conditions were used to incubate spermatozoa and oviductal epithelial cells in the presence of six hexose sugars at concentrations of 0, 2, 10 and 50 mmol l(-1). A significant (P < or = 0.05) reduction in the binding of spermatozoa to the oviductal epithelium was detected with 2, 10 and 50 mmol maltose l(-1), 50 mmol lactose l(-1) and 50 mmol mannose l(-1). These findings support the hypothesis that attachment of pig spermatozoa to oviductal epithelium before fertilization is mediated by carbohydrate recognition.
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The sensitive mRNA phenotyping technique of reverse transcription–polymerase chain reaction was used to demonstrate that insulin receptor mRNA is present in rat embryos during the preimplantation period. In addition, mRNA encoding insulin-like growth factor (IGF) type I and type II receptors have also been detected in rat preimplantation embryos. IGF-I mRNA was not detected in preimplantation embryos but was found in oviducts and uteri of prepubertal and early pregnant rats. IGF-II mRNA was present in both embryos and in oviducts and uteri during the preimplantation period. These findings suggest that insulin and IGF-I could influence early embryo development in endocrine or in paracrine fashions, whereas IGF-II may have an additional autocrine mode of action in affecting preimplantation embryos in rats.
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Summary. The cumulus oophorus surrounding eggs from C57BL/6 mice was digested by bovine or leech hyaluronidase significantly more rapidly than that surrounding eggs from BALB/c mice. The zona pellucida of C57BL/6 eggs was also more rapidly attacked by pronase. Three other sublines of C57BL showed the same characteristics. Measurements of susceptibility to hyaluronidase and pronase on eggs from the CXB recombinant inbred strains indicated that variation at a minimum of 2 loci affected each character. The lack of correlation between susceptibilities to the 2 enzymes across the recombinant strains implied that these differences separately affect the substrates of the enzymes, rather than reflecting a common difference in the process of oocyte maturation. The variation in susceptibility was unrelated to differences, controlled by the Ped and Qa-2 loci, in the rate of later embryonic cleavage. However, pronase susceptibility was significantly correlated with the early onset of the first cleavage.
Keywords: cumulus; zona pellucida; inbred strains; fertilization; cleavage; mouse
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A preparation for the maintenance of human Fallopian tubal epithelial cells as a polarized layer in primary culture was used to study the electrophysiological basis of tubal fluid formation in terms of the movement of Na+, K+ and CI− ions. Transepithelial potential difference (PD) and short-circuit current I scc) were recorded by mounting the epithelial cells in a modified Ussing chamber. Resistance (R) was calculated from the measurements of PD and I scc. The epithelia, although confluent, formed a 'leaky' electrical system and resistances greater than 300 Ω cm−2 were rarely achieved. The sodium channel blocker, amiloride (100 μmol l−1), produced only small effects on PD and I scc. The potassium channel blocker, tetraethylammonium chloride (TEA) (25 mmol l−1), also produced small, but significant changes in PD, I scc and R while the chloride channel blocker, 4-acetamido-4′-isothiocyanostilbene-2.2′-disulfonic acid (SITS) (1 mmol l−1), induced a marked increase in PD and I scc, and a fall in R, when added to the basal surface of the cells. Bathing the apical surface of the cells with chloride-free medium also produced a marked increase in PD, I scc and R; bathing the basal surface of the cells with chloride-free medium produced a marked decrease in PD and I scc. Extracellular ATP (10 μmol l−1 added to either the apical or the basal surface of the cells, induced a transient increase in PD and I scc and a decrease in R. Amiloride, TEA or furosemide had no effect on the response of the cells to ATP. SITS, applied to the apical surface, significantly reduced the response of the cells to ATP. We conclude that the major driving force for human tubal fluid formation is the transepithelial secretion of chloride ions into the oviduct lumen and that exogenous ATP is a potential modulator of secretion.
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Blastocyst formation is dependent on the differentiation of a transporting epithelium, the trophectoderm, which is coordinated by the embryonic expression and cell adhesive properties of E-cadherin. The trophectoderm shares differentiative characteristics with all epithelial tissues, including E-cadherin-mediated cell adhesion, tight junction formation, and polarized distribution of intramembrane proteins, including the Na–K ATPase. The present study was conducted to characterize the mRNA expression and distribution of polypeptides encoding E-cadherin, β-catenin, and the tight junction associated protein, zonula occludens protein 1, in pre-attachment bovine embryos, in vitro. Immunocytochemistry and gene specific reverse transcription–polymerase chain reaction methods were used. Transcripts for E-cadherin and β-catenin were detected in embryos of all stages throughout pre-attachment development. Immunocytochemistry revealed E-cadherin and β-catenin polypeptides evenly distributed around the cell margins of one-cell zygotes and cleavage stage embryos. In the morula, detection of these proteins diminished in the free apical surface of outer blastomeres. E-cadherin and β-catenin became restricted to the basolateral membranes of trophectoderm cells of the blastocyst, while maintaining apolar distributions in the inner cell mass. Zonula occludens protein 1 immunoreactivity was undetectable until the morula stage and first appeared as punctate points between the outer cells. In the blastocyst, zonula occludens protein 1 was localized as a continuous ring at the apical points of trophectoderm cell contact and was undetectable in the inner cell mass. These results illustrate that the gene products encoding E-cadherin, β-catenin and zonula occludens protein 1 are expressed and maintain cellular distribution patterns consistent with their predicted roles in mediating trophectoderm differentiation in in vitro produced bovine embryos.
