The termination of delayed implantation in the fur seal (Daniel, 1971,1974), mouse (Aitken, 1977), rat (Surani, 1975) and roe deer (Aitken, 1974,1975) is thought to be associated with the oestrogen-induced release of a protein-rich endometrial secretion into the uterine lumen. Finn (1974), however, observed that the intraperitoneal injection of actinomycin D, an inhibitor of protein synthesis, terminated delayed implantation in the mouse within 48–72 h. It was suggested that actinomycin D released the blastocysts from their state of diapause by inhibiting the transcription of a proteinaceous inhibitor of blastocyst attachment (Finn, 1974). Actinomycin D may, however, have increased the protein content of the uterine lumen either through the 'superinduction' of uterine proteins (Tomkins et al., 1969) or, possibly, through the stress-induced release of steroids from the adrenal glands. A study was therefore designed to determine the influence of actinomycin D on uterine secretory activity in the mouse.
R. J. Aitken
School of Veterinary Medicine, University of Cambridge, U.K.
The roe deer is the only ungulate known to exhibit the phenomenon of delayed implantation. Shortly after ovulation in early August, the blastocyst loses its zona pellucida and enters a 5-month period of diapause during which the trophoblast exhibits a minimum of mitotic activity and the inner cell mass remains undifferentiated. Delayed implantation is terminated in early January by the sudden, rapid elongation of the trophoblast and subsequent placental attachment (Short & Hay, 1966). The resumption of rapid embryonic growth appears to coincide with the release of a secretion from the endometrial glands (Aitken et al., 1973; Aitken, 1975). This communication presents the results of an analysis of the carbohydrate content of this secretion.
Eighty-five roe deer shot during the annual Forestry Commission cull at Thetford Chase, Norfolk, provided the material for this study. The uterus was removed from each animal immediately
R. J. AITKEN
The concentration of zinc in the endometrium of the roe deer was maintained at a constantly high level throughout delayed implantation and at the time of rapid embryonic growth. By contrast, the zinc content of the uterine flushings was uniformly low and often undetectable (<0·1 μg/ml).
The concentration of calcium in the endometrium showed a highly significant (P<0·001) increase during delayed implantation reaching maximal levels upon the resumption of rapid embryonic growth. The calcium content of the uterine flushings showed a small but significant (P<0·001) rise in the late stages of diapause followed by a further marked increase during the rapid elongation of the blastocyst in January.
R. J. AITKEN
The results presented in this paper indicate that delayed implantation in the roe deer is due to a lack of certain essential factors which are needed to induce and support the process of embryonic growth. These factors are eventually supplied, in the first weeks of January, as a secretion emanating from the endometrial glands. This secretion contains uterine-specific and serum proteins, about twenty free amino acids, protein-bound glucose and galactose and, rather surprisingly, a free ketose which appears to be fructose. Elongation of the roe deer blastocyst is also correlated with a rise in the concentration of plasma oestrogens, an endocrine change that may stimulate the endometrial glands into secretory activity. However, since simultaneous changes were not observed in the ovaries, the elevated oestrogen levels may be a consequence rather than a cause of embryonic growth.
R. J. Aitken
In this article I shall be discussing our state of knowledge with respect to the composition and function of tubal and uterine secretions with particular reference to the possibilities for contraceptive attack. Before considering the secretions themselves it is important to establish the type of contraceptive we are hoping to develop as a result of these studies. One possible approach might be to search for specific proteins or glycoproteins in the genital tract secretions which are essential for the continuation of early embryonic development and which might, therefore, be used as targets for the immunological suppression of fertility. In both the tubal (Moghissi, 1970) and uterine (Roberts, Parker & Henderson, 1976; Maathuis & Aitken, 1978a, b) secretions of the human genital tract biochemical evidence exists for the presence of nonserum, possibly unique, proteins (Pl. 1, Fig. 1). The specificity of these proteins has not yet been established immunologically, however, and we may do well to remember that the protein "uteroglobin" (Krishnan & Daniel, 1967; Beier, 1968) was thought to be a specific component of rabbit uterine secretions for a decade until it was also discovered in the lungs, tubal secretions and seminal plasma of this species (Noske & Feigelson, 1976). Our uncertainty concerning the specificity of uterine and tubal proteins and the attendant hazards of autoimmune disease (Tung, 1976) should perhaps discourage us from this approach. An alternative route towards the immunological suppression of fertility might be to search for specific embryonic factors which are responsible for regulating the synthesis or release of critical components in the oviductal or uterine secretions. The advantage of this approach is that the target antigen is of embryonic origin and would, therefore, only be exposed to the maternal immune system for a limited period of time. The specificity of the embryonic factors involved would still have to be demonstrated however. Alternatively we might try to regulate the synthesis or release of critical secretory components by pharmacological means, using, for example, anti-oestrogens or antiprogestagens. Common to both the immunological and pharmacological approaches is the need to identify processes in early development in which tubal or uterine secretions play an important part. In the following sections I shall consider the possible roles played by these secretions and assemble the evidence for an embryonic or pharmacological influence on their composition.
R. J. Aitken
Summary. The protein content of the mouse uterine lumen increased significantly (P < 0·001) on Day 4 of pregnancy, the day of implantation. This increase was associated with the presence of 14 serum and 22 non-serum proteins in the lumen; the major serum proteins were classed as high molecular weight slow α-globulins, while the dominant non-serum components consisted of slow and fast α-globulins, 6 prealbumins and a large quantity of proteinaceous material migrating near the origin of the gels.
During experimental and lactational delayed implantation the protein levels were constantly low, transferrin, haemoglobin and albumin dominating the protein pattern. After administration of oestradiol-17β, however, a biphasic uterine response was detected, significant increases in luminal protein concentration being observed within 12 h and again at 40–48 h after injection. The first phase of this response involved an influx of serum and non-serum proteins into the uterine lumen, most proteins migrating as high molecular weight slow α-globulins. The second phase involved an increase in the intensity of many non-serum components, the major proteins having Ra values of 0·06,0·10 and 0·32. The qualitative, but not the quantitative, aspects of this response to oestradiol were identical in the absence of blastocysts.
R. J. Aitken
A biochemical study of the mouse uterus during normal pregnancy (Aitken, 1977) revealed qualitative and quantitative changes in the protein content of the uterine flushings at the time of blastocyst activation and implantation. The purpose of the present study was to determine the nature of the changes taking place during the course of pseudopregnancy.
R. J. AITKEN
According to the Lyon hypothesis (Lyon, 1961, 1972), random genetic inactivation of one of the two X chromosomes occurs in the somatic cells of female mammals at an early stage of embryonic development. Certain properties of this inactivated chromosome, such as its late replication during the S-phase of mitosis (Taylor, 1960; Grumbach & Morishima, 1962; Mukherjee & Sinha, 1963), heteropyknosis during prophase and the formation of a sex chromatin body during interphase (Ohno, Kaplan & Kinosita, 1959; Ohno & Hauschka, 1960) have been used as criteria for establishing when the process of inactivation begins. These features first appear in blastocysts at about the time of implantation in the cat (Austin & Amoroso, 1957), dog (Austin, 1966), rat (Zybina, 1960), hamster (Hill & Yunis, 1967), vole (Microtus agrestis) (Lee & Yunis, 1971), rhesus
R. J. Aitken and R. A. Elton
Summary. A Poisson–gamma distribution model has been developed for studying sperm–oocyte interaction in the zona-free hamster egg penetration test. With the aid of this model we have analysed the relationship between the penetration results achieved with semen of normal fertile men and the concentration of spermatozoa and oocytes used in the assay. From this analysis we have drawn up reference tables estimating the number of spermatozoa and occytes which must be present in the assay for the result to represent a significant (P < 0·05) decline in sperm function relative to the normal fertile population.
Since there is, as yet, no fixed protocol for the penetration assay, these tables have been constructed to fit a range of methodologies varying with respect to the nature of the preincubation phase and including (a) a 7-h preincubation in Medium BWW, (b) a 3-h preincubation in hyperosmotic Medium BWW or (c) a 3-h preincubation in the presence of A23187.
R. J. Aitken and R. A. Elton
Summary. The value of Poisson distribution theory in describing and predicting the nature of sperm—egg interaction in vitro has been investigated using an interspecific in-vitro fertilization system, incorporating zona-free hamster oocytes and human spermatozoa. The frequency distribution of polyspermic oocyte penetrations in 72 experiments exhibited good agreement with the Poisson distribution at all levels of fertilization, indicating that each oocyte must be of equal penetrability and that there can be no block to polyspermy in this interspecific system. Poisson distribution theory also accurately described the relationship between oocyte penetration and sperm motility in 50 out of 54 separate experiments spread across 10 serial dilution curves. For each dilution series the shape of the fitted curve was fixed but its location along the x-axis varied from donor to donor. The fixed nature of the relationship between sperm motility and egg penetration enables the results of such in-vitro fertilization experiments to be corrected for the number of motile spermatozoa in the incubation media. On the basis of these findings a protocol is described for assessing the results of the zona-free hamster oocyte penetration assay, which involves analysis of the degree of polyspermy followed by the application of Poisson distribution theory to correct the results to a standard concentration of motile spermatozoa. Changes in the penetrating ability of human spermatozoa after vasectomy and characterization of the degree of inter-ejaculate variation in penetrating potential are two clinical examples of such analyses given in the text. The statistical methods described in this paper should also be of general relevance to the study of fertilization mechanisms, in providing a rationale by which to analyse the quantitative nature of sperm—egg interaction in vitro.