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
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
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
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
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
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
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 John Aitken
There has never been a greater need for scientists trained in reproductive science. Most developed countries are witnessing unprecedented rates of recourse to assisted conception sitting cheek-by-jowl with high rates of induced abortion. This article addresses these two incongruous faces of reproductive healthcare. Every year at least 44 million abortions are performed worldwide, many under unsafe and insanitary conditions that carry a significant risk to the lives of women deprived of safe, effective methods for controlling their fertility. Although birth control is a complex issue involving myriad social and political factors, the technical vacuum in this area is significant. Through no fault of the family planning authorities, there have been no radically new methods of fertility control since the oral contraceptive pill was introduced in 1960 and even this contribution to planned parenthood has its roots in the biochemistry of the 1920s and 1930s. Moreover, the pharmaceutical industry has, by and large, turned its back on fundamental research activities in this area. At present, our major investment in reproductive healthcare involves treating ever-increasing numbers of couples with assisted reproductive technologies (ART). However, these treatments are often delivered without critically considering the underlying causes of this condition or seriously contemplating the long-term consequences of the current enthusiasm for such therapy. Significantly, the clinical factors underpinning the commitment of couples to ART include advanced maternal age and a variety of lifestyle factors, such as smoking and obesity, which are known to compromise the developmental potential of the oocyte and DNA integrity in spermatozoa.
R. John Aitken
A great deal of evidence has accumulated in recent years to suggest that there has been a gradual increase in male reproductive pathology over the past 30–40 years, as evidenced by increased rates of testicular cancer and declining semen quality. The hypothesis is advanced that this phenomenon is causally related to the ability of male germ cells to generate reactive oxygen metabolites. When produced in low levels, such metabolites are thought to enhance sperm function by stimulating DNA compaction and promoting a redox-regulated cAMP-mediated pathway that is central to the induction of sperm capacitation. When produced in excessive amounts, the same metabolites stimulate DNA fragmentation and a loss of sperm function associated with peroxidative damage to the sperm plasma membrane. Free radical-induced mutations in the male germ line may also be involved in the aetiology of childhood cancer and recent increases in the incidence of seminoma. In light of these considerations, establishing the mechanisms for free radical generation by the male germ line and determining the factors that influence this activity are important objectives for future research in this area.
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.