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Embryo transfer surgery is an essential step in the process of generating gene-modified mice, regardless of whether the embryos were modified by DNA, RNA CRISPR components, or embryonic stem cells, or whether they were microinjected or electroporated. Transfer is also a necessary step for assisted reproductive techniques such as rederivation and reanimation by in vitro fertilization. The manipulated embryos must be returned to the reproductive tract of a pseudopregnant recipient female mouse, wherein the transplanted embryos develop to term. Embryos may be transferred to either the oviduct or uterine horn, depending upon the developmental status of the embryos and the stage of the recipient. This chapter will describe the process of transferring embryos surgically to a recipient female.

In litter bearing mammals the intrauterine position of a fetus with respect to other fetuses in each of the uterine horns has profound effects on its later morphology, physiology, and behavior. This effect was first reported in rats (Clemens et al., 1978). Similar work on mice has produced a considerable knowledge concerning intrauterine position effects (vom Saal and Bronson, 1978 vom Saal and Bronson, 1980; reviews by vom Saal, 1989; vom Saal et al., 1992). Sexual differentiation of female mouse fetuses that are positioned between two males is affected by androgen from the neighboring male fetuses (vom Saal and Dhar, 1992). Females that are located in utero between two males are designated as from the 2M position, those located between two females are designated as from the 0M position (or these are sometimes referred to as 2F animals), and females positioned in utero between a male and a female are designated as 1M females. A similar terminology can be applied to males with respect to their in utero position in relation to other males.

Implantation implies major cell modifications of the trophoblast and the uterine endometrium. These cellular changes render the two tissues receptive and allow an intimate adhesion giving rise to a functional placenta. Classically the process of implantation is subdivided into 4 main steps: a pre-attachment period; apposition and adhesion of the blastocyst followed by invasion of the uterine endometrium (SCHLAFKE and ENDERS, 1975). In artyodactyls this division of implantation applies partly, with some specific characteristics. There is a long pre-attachment period (2–3 weeks), a diffuse and progressive attachment of the trophoblast along the uterine horns and a non-invasive phase of adhesion.

In most mammals studied, the embryo starts developing in the oviduct and about the time it enters the uterus it is constituted by a few blastomeres rarely exceeding 12 to 16 in number. It is hard to believe at first sight that such minute biologic entity may have to signal its presence to insure its survival at the same time that its few cells are so busy executing a progressively complex developmental process.

Anomalies of the uterus may be interpreted as arrested development. The various forms may be related to: (1) agenesis or inadequate differentiation or regression of the uterine part of one or both miillerian ducts (2) abnormalities in fusion of the miillerian ducts (3) abnormalities in involution of the septum after fusion of the miillerian ducts (4) retarded growth and development of the normally differentiated organ

Histological studies were done by light and electron microscopy to improve biological tests used to evaluate the pharmacological properties of RU 486. The compound prevented the progesterone-induced appearance of giant mitochondria in uterine glandular cells in ovariectomized rats and did not show any progestogen-mimetic effect. Its antiprogestational activity was demonstrated by abortion in rats where RU 486-induced changes in the deciduoma cells could be observed, even prior to the changes caused by ovariectomy. The hypertrophic effect of RU 486 on the uterus of immature animals was distinguished from the hyperplastic effect of estradiol.

Attempts were made to visualise the cyclic behaviour of the lymph vessels in the uterus of animals which had been pregnant, using Patent blue violet and Japanese ink in a modified form adapted to the natural absorption possibilities of the uterus. Here also the mouse, an animal with a haemoendothelial type of placenta, is a particularly suitable experimental animal, because material can be obtained in a known phase without great expense of material and time.

The kininogenase activity of tonin has been demonstrated by Ikeda and Arakawa, 1984. Tonin of the rat submandibular gland contracts the rat uterus independent of addition of the substrate. On repetition, the same dose of enzyme elicited desensitization. When a double dose was used the contraction again occurred. After desensitization to tonin the contraction to kallikrein was reduced about 80% of the control. The desensitization to kallikrein lightly reduced the contraction to tonin. When the muscle was desensitized to trypsin tonin did not evoke contraction. These experiments suggest the presence of two different substrates in the uterus, one more specific to kallikrein and the other for tonin.

Uterine muscular activity is influenced by ovarian steroid hormones, by nervous activity, and by endogenous, humoral or locally produced, uterus-activating agents such as the neurohypophyseal peptides, the prostaglandins, and various biogenic amines. Purely physical factors, such as stretch, also play a part in the regulation of myometrial function. While the neural control of uterine function is more intimately linked to uterine vascular supply than to its muscular elements, at least in rats and rabbits, the hormonal factors exert a profound influence directly on the contractile components of the uterus. However, in spite of much progress in recent years in the understanding of the mechanism of action of steroid hormones, the integration of biochemical events at the molecular level into various contractile responses at the tissue level remains to be elucidated. The possible interrelationship between endocrine factors and the nervous system in integrating the total activity of the whole organ has also to be clarified, but all evidence points to the hormonal and humoral agents as the dominating factors in the regulation of uterine contractility.

In order to determine the possible role of estrone (E1) formation during the implantation process, estradiol-17)β dehydrogenase (E2DH) activity was investigated in rat endometrium during the estrous cycle, the first days of pregnancy, and in castrated rats treated with estradiol (E2) alone or in combination with progesterone (P). Endometrium of mature Sprague-Dawley rats was removed from the myometrium by gentle scraping of longitudinally opened uterine horns. E2DH assays were performed on endometrial homogenate according to the method of Tseng and Gurpide (1974) (E2DH in pmol/mg protein ·hr).

In rats and mice, the uterine surface epithelium on Day 5 of pregnancy appears still intact while the decidual reaction is already spreading (Finn, 1977). Therefore the physiological signal from the blastocyst that initiates decidualization must be conveyed in some way through the epithelial layer. Uterine trauma, such as scratching or crushing, can elicit a decidual cell reaction (DCR) in pseudopregnant or adequately sensitized spayed animals, but whereas a “normal” DCR requires previous intervention of small amounts of estrogen (Finn, 1977), traumatic deciduomas can also be obtained with progesterone alone (De Feo, 1967). It has therefore been argued that luteal estrogen primarily acts upon epithelial cells by enabling them to transmit the embryonic message, whereas trauma would bypass this step and act on the stroma directly (Finn and Martin, 1974). Another opinion holds that the epithelium is a compulsory intermediate in any type of DCR, for traumatic procedures would be efficient only if they also damage epithelial cells (Psychoyos, 1974; Thibault, 1978).

Numerous reports have appeared dealing with various molecular components of uterine fluid during implantation or delayed implantation (Pinsker et al., 1974; Aitken, 1977; Surani, 1977; Hoversland and Weitlauf, 1978) and the findings have led to the suggestion that components of uterine fluid influence or even regulate metabolism in preimplantation embryos. In those experiments, uterine fluid was usually collected by flushing buffer through the uterus. The resulting fluids were analyzed for the total amount of various components, or the ratio of those components to protein. One difficulty in interpreting such data is that the concentration of the various substances in situ cannot be determined without an estimate of the original volume of uterine fluid. To resolve this difficulty, the present experiments were undertaken to determine the volume of uterine fluid in mice under various endocrine regimens. The method used was a modification of the technique described by Kulangara (1972).

Oxygen is essential for blastocyst survival. Since, prior to implantation, the blastocyst has no direct connection with the maternal vasculature, a supply of oxygen sufficient to meet its metabolic needs must be available within the uterine lumen. In vitro studies indicate that mouse blastocysts require at least 7 mmHg of oxygen to sustain development (Auerbach and Brinster, 1968). While in vitro experiments have provided considerable information concerning conceptus metabolism and development, the environmental conditions to which the blastocyst is subjected during culture are different from the environment provided by the uterine lumen. Thus, until in vivo conditions are determined, the extent to which in vitro environments duplicate that in which blastocyst development and implantation normally occur, remains unknown.

Many types of mechanical manipulation of the mammalian conceptus can result in embryonic death, growth retardation, or malformation. Crushing the embryonic site results in death and provides a simple way to control the number and intrauterine positions of conceptuses (Jensh et al, 1970; Barr, 1970, 1971). Reduction of amniotic fluid by amniocentesis may result in malformation or fetal death (Trasler et al., 1956; Adams, 1960; DeMyer and Baird, 1969). Direct surgical manipulation of the fetus may result in death or specific malformation of the operated organs (Hall and Schneiderhan, 1945; Wells, 1950; DeMyer and Baird, 1973). Surgical approaches to the mammalian embryo in vivo have not been highly successful, and surgery on the fetus, while possible in small animals, has been most widely used in large animals such as sheep and dogs. Most of this discussion of surgical techniques in teratology will be concerned with the rat since it is with this animal that we are most familiar. The same techniques can, however be applied to other rodent species with appropriate modifications.

Revadi and Thomas (1979) have established the existence of normal cyclic variations in the human uterine endocervical mucosa. Their further studies (1980) on endogenously-produced oestrogen states and the human uterine endocervix showed that oestrogen produces a progressive increase in endocervical mucosal cell heights which is unrivalled by the endocervix, even in the pregnant state. On the basis of these studies, they hypothesized, that though oestrogen, was probably responsible for the secretion of excessive quantities of endocervical mucus, the release of this mucus was probably mediated by progesterone. In their studies basal cell proliferation was never seen.

Implantation involves changes in all cell types of the uterine endometrium in preparation for attachment of the blastocyst to the uterine wall. The artificially stimulated decidual cell reaction (DCR) has been used as a model system for studying the uterine responses to implantation (Finn and Porter, 1975; Classer and Clark, 1975). In the decidual cell reaction, uterine stromal cells proliferate and are transformed into decidual cells. The hormonal requirements for the oil-stimulated DCR closely approximate the hormonal preparation of the uterus at the time of natural implantation. The principal advantages offered by the artificially stimulated DCR in hormone-primed ovariectomized animals include (a) a more extensive reaction than that occurring in natural implantation; (b) a more precise time course of the reaction, particularly short times after stimulation; (c) the ability to study the roles of estrogen and progesterone in the reaction; and (d) the ability to use large numbers of animals in a single experiment.

The transformation of uterine stromal cells into decidual cells is an integral part of normal implantation (Finn and Porter, 1975). The decidual cell reaction (DCR) can be induced in a uterine horn of a hormone-primed ovariectomized mouse by intralumenal injection of sesame oil or by crushing the uterine horn (trauma). Traumatic stimulation does not require the small “nidatory” dose of estradiol a few hours prior to the stimulus; this is required for oil induction of the response just as it is required for natural implantation. Either stimulus leads to the transformation of uterine stromal cells into rapidly proliferating decidual cells. Decidual cells are not histologically identifiable until 24 hr after stimulation. However, various biochemical and physiological changes precede this transformation and can be quantitated at early times following artificial stimulation of the DCR.

Sheep (Ovis aries) that are not pregnant have recurring estrous cycles of about 17 days’ duration during the breeding season, i.e., August through December to January. The first day of behavioral estrus is designated Day 0 with ovulation occurring about 30h after onset of estrus and the ovulatory surge of Luteinizing Hormone (LH). The estrous cycle is characterized by growth (Days 4–12) and then regression (luteolysis) of the corpus luteum (CL). Luteolysis begins on about Day 15 after onset of estrus. Prostaglandin F2-alpha (PGF), produced by the uterine endometrium, is the uterine luteolytic hormone in sheep (1). Beginning on Days 14 to 15 and continuing until the onset of the next period of estrus, PGF is released in a pulsatile manner. About 5 episodes of PGF release per 24h are required for luteolysis (2,3).

Nerve—muscle preparations of other smooth muscles in the genito-urinary tract have been studied less extensively than those of the vas deferens. One reason is that, unlike the vas deferens, these other muscles usually show considerable spontaneous, myogenic activity. It is assumed that neural influences modulate rather than initiate this activity so that the effects of nerve stimulation are more subtle and therefore more difficult to analyze than in the vas deferens. Another reason is that the innervation density, determined histochemically and biochemically (Labay et al., 1970; Marshall, 1970), is considerably less in genito-urinary muscles other than the vas deferens. Thus the electrophysiological investigations of neuroeff’ector transmission at the cellular level are often more ambiguous and less predictable in the former muscles. Nevertheless, studies on nerve-muscle preparations from a variety of smooth muscles are of importance not only from a comparative pharmacological viewpoint, but also as a contribution to a better understanding of the role of the nervous system in the control and regulation of smooth muscle in general.

Regulation of fertile or nonfertile reproductive cycles in mammals depends on interaction of ovarian, uterine, pituitary, and CNS function and exteroceptive stimuli. These cycles can be modified by a variety of actions such as mating, presence of a conceptus or conceptus secretory products, cervical stimulation, endometrial abrasion, and complete or partial absence of the uterus. An essential feature in this regulation is the functional status of the ovary, particularly that of the corpus luteum. It is of particular interest to elucidate the factors that affect the development, maintenance, and regression of the corpus luteum and the role of the uterus in these events. The mammalian uterus has a very profound effect on luteal function. The presence of the uterus alters the process of regression and morphological demise of the corpora lutea. This chapter deals with uterine-ovarian functions in several species and with how uterine-ovarian interactions occur to modify ovarian function during cyclicity and pregnancy.

Glandular kallikrein (EC.3.4.21.8) releases lisyl-bradykinin (kallidin) from kininogen substrates by limited proteolysis (Chao et al, 1981). The responses of the smooth muscle to kallikrein are attributed to kinin formation and a subsequent peptide-receptor interaction (Barabe et al,1977; Fritz et al, 1979; Odya and Goodfriend, 1979). Beraldo et al.(1966) reported that rat urinary kallikrein induced rat uterine contractions in the absence of added kininogen. Nustad and Pierce (1974) suggested that uterine contractions elicited by rat urinary kallikrein depend on kinin liberation from some uterine kininogen. Beraldo et al.(1976) demonstrated the presence of kininogen in the rat uterus. Chao et al.(1981) reported that rat glandular kallikrein can cause the contraction of isolated rat uterus independent on kinin formation. As can be seen, the mechanism of kallikrein-induced uterine contraction was uncertain. We used pure rat submandibular gland kallikrein in order to explain this mechanism.

Two series of experiments have been conducted to identify possible target tissues for relaxin in the rat (Cheah and Sherwood, 1980). This paper will briefly describe the results of these experiments in which we: (a) examined the distribution of radioactivity in various tissues following the injection of 125I- labeled relaxin (125I-relaxin) in vivo and (b) measured the cAMP levels in tissues following incubation with relaxin in vitro.

The rapid regression of the bovine corpus luteum at the 17th–19th day of the estrous cycle requires the presence of the uterus and appears to be mediated by estrogen and one or more uterine luteolysins acting locally on the ipsilateral corpus luteum (1,2). Little information is available as to how the presence of a viable embryo in the uterus causes maintenance of the bovine corpus luteum. Del Campo et al. (3) suggested a local relationship between a gravid uterine horn and the adjacent ovary in the pregnant cow. Although the corpus luteum is necessary for maintenance of pregnancy in the cow, at least until Day 236 (4), the transition of the cyclic corpus luteum to the corpus luteum of pregnancy occurs before the embryo implants. Staples et al. (5) showed that very rapid elongation of the trophoblast occurs at about Day 15, just prior to the time of cyclic corpus luteum involution. Mesoderm formation also begins at Day 15, and the bi-nucleated giant cells of the trophoblast (6) first appear at this time (5). Greenstein et al. (7) suggested that these cells may play a role in stimulating maintenance of the corpus luteum of pregnancy. These facts suggest that the developing embryo either inactivates the normal uterine luteolytic mechanism, or produces a potent luteotrophin that overcomes this mechanism.

The embryonic signal that initiates luteal maintenance occurs by about Day 12 in the sow (Dhindsa and Dzuik, 1968), Day 13 in the ewe (Moor and Rowson, 1966), and Day 16 in the cow (Betteridge et al., 1978). The mechanism responsible for maintenance of a corpus luteum (CL) during early pregnancy, and in particular the way in which the embryo influences this process, is not clearly understood but may involve a local affect of the conceptus on utero-ovarian blood flow. Data presented by Ford et al. (1976) demonstrated a preferential effect of the ovine and bovine conceptus for reducing in vitro constriction of the uterine artery supplying the gravid horn on Day 15 and Day 17 postcoitus, respectively. Transient increases in blood flow to gravid uterine horns of ewes have been observed on Days 13 to 15 of pregnancy (Greiss and Anderson, 1970).

The number of spermatozoa in the uterine tubes of the sow varies between 101−104, debending on the amount of semen deposited and the efficiency of the sperm transport (Alanko, 1968).

Pseudopregnancy can be induced by a number of drugs (see Meites et al., 1963), among which brain catecholamine depletors as reserpine or or-methyl-dopa (Ratner et al., 1965; Coppola et al., 1965). This led to the assumption that brain catecholamines might be obligatory for the synthesis or release of the hypothalamic prolactin-inhibiting factor (PIF) (Coppola et al., 1965). In fact, in the ventral portion of the hypothalamus a high content of catecholamines could be demonstrated by Carlsson et al. (1962) and Fuxe (1964) by means of a histochemical fluorescence technique.

Zipper et al.(1969) reported that a small copper (or zinc) loop placed in the lower portion of one of the uterine horns in rabbits produced a decrease in the number of implantation sites without producing a significant alteration in the control uterus. Silver, tin and magnesium were not effective. Zipper and co-workers (1970) utilized this observation to design a copper-containing intrauterine device for the prevention of fertility in the human female and reported that clinical effectiveness was related to the amount of copper present (Table 1).

Ovulation and corpus luteum formation recur at approximately 16-day intervals in the guinea-pig, at a time when regression of the previous set of corpora lutea is marked. Loeb (1911a, b) recorded that the corpora lutea are fully developed from the histological point of view from the sixth day after ovulation, and that regressive changes can be detected on the tenth day. When the changes in the volume of the corpora lutea during the cycle were traced by Rowlands (1956), Perry and Rowlands (1962a) and Bland and Donovan (1966a), it was found that the maximum size is reached at about the eleventh or twelfth days (Figure 1). Thereafter regression sets in and is rapid. It is perhaps unfortunate that the morphological changes in the corpora lutea may not be correlated with their functional state for Rowlands and Short (1959) found that the concentration of progesterone in luteal tissue 11–13 days after ovulation (7·7 µg/g) is half that at 6 days (16·2 µg/g) despite the lack of histological modification in the interval.

The preimplantation period consists of two phases, tubal and uterine, and although this communication is primarily concerned with the uterine phase, some reference must be made to the former since the two are so intimately connected. Moreover, so far as the early embryo and uterine environment are concerned, the most significant change in ovarian function, that from oestrogen to progesterone dominance, takes place whilst the egg is still in the Fallopian tube.

One of our contemporary authorities on the physiology of the uterus, Reynolds (1949), quoted an English physician, Tyler Smith, who wrote in the Lancet of 1848 that the uterus ‘supports the race in the same way that the stomach and heart support the individual’. As the uterus is the obvious organ to reproduce young or to develop embryos, the historical and cultural aspects of the uterus can be traced back to very ancient times and to the very primitive tribes (Plaut, 1959). Its biological importance and its physiological function have been dealt with by Marshall in the second edition of The Physiology of Reproduction (1922), and in the third edition by Parkes (1952, 1956 and 1960), also by Alan, Danforth and Doisy in the second edition of Sex and Internal Secretions (1939) and the third edition by Young (1961). One important function of the uterus has been rather neglected, however, that it also supports spermatozoa for a certain length of time. This function is perhaps just as important as that it ‘supports the race’ for generations or supports the embryos for several months. I shall deal with the reaction of the uterus on spermatozoa and on early eggs by reporting some of my own observations and the contributions of other learned colleagues in the field of animal reproduction.

When we found that it is possible to transplant part the the uterine horns from one side to the other in the same animal, we began to transplant the uterine horn from one animal to another.

At our Clinic we were already previously concerned with the problem of the possible transplantation of female sex organs, as apparent from the lectures and papers of one of our collaborators on the experimental transplantation of the pituitary, tubera cinerea and ovaries. In recent years we rather reflected on the transplantation of the ovaries and oviducts. These operations could be of therapeutic use in irreparable obstructions of the oviducts or conditions after bilateral salpingectomy.