11

Reproduction

Franklin H. Bronson, Charles P. Dagg, and George D. Snell

This chapter is a partial revision of a chapter in Biology of the Laboratory Mouse ( Snell, 1941). A few of the previously published sections — sexual maturity, estrous cycle, mating, and gestation — have been rewritten. The remaining sections have been altered only slightly. Only references actually dealing with laboratory mice have been included and, particularly in the rewritten sections, emphasis has been placed on the descriptive details of normal reproduction (e.g., time and magnitude characteristics). More comprehensive texts (such as Young, 1961) should be consulted for the general principles of reproductive physiology.

SEXUAL MATURITY

Reproduction in the mature female mouse consists of a series of related and dependent hormonal and neural events which function to insure the successful production of new members of the species. Basic in this process is the interaction of the anterior pituitary, placental, and gonadal hormones. Also basic is the role of the central nervous system, particularly the hypothalamus, in regulating hormone release by the anterior pituitary and, in turn, the effects of gonadal hormones on the hypothalamus itself. Follicle stimulating hormone (FSH), a gonadotropin released by the anterior pituitary, acts primarily to promote gametogenesis in both sexes. Luteinizing hormone (LH), another gonadotropin, has a primary function of promoting secretion of the gonadal hormones; estrogens and progesterone in the female and androgen in the male. A third anterior pituitary hormone, prolactin, functions in lactation and the development of the ovary during pregnancy. The gonadal hormones have the responsibilities of maintenance of secondary sexual characteristics and of the proper condition of the reproductive tract as well as acting on the central nervous system to insure successful mating. Full sexual maturity depends on a delicate and as yet incompletely understood interplay among all of these components and the maturation of each proceeds at a somewhat different rate.

At birth the mouse hypophysis is physiologically undifferentiated from the gonadotropic viewpoint ( Vivien, 1950) and, in addition, the ovaries are unresponsive to gonadotropin ( Francke, 1948). Sex differentiation of the hypophysis is usually realized by day 6 in males, and before day 12 in females. Ovarian weight becomes responsive to exogenous FSH by days 6 to 9, but follicle size cannot be altered by FSH injections much before 12 to 15 days of age ( Ben-or, 1963). Antrum formation normally begins in some follicles by days 12 to 14.

The threshold of ovarian (follicle) responsiveness decreases following initiation of competence to a low at about 3 weeks of age and, thereafter, is elevated to a relatively mature level by 30 days of age. Zarrow and Wilson ( 1961), after injections of pregnant mare serum (PMS), a hormone preparation having activity similar to FSH, and human chorionic gonadotropin (HCG; LH-like in activity), found the follicles competent to respond by about 14 days of age. Ovulation occurred in 40 per cent of the females at 13 to 14 days of age; 69 per cent ovulated by 15 to 18 days; 80 per cent by 19 to 20 days; and about 86 to 100 per cent thereafter. Maximum ova counts ran from 3.6 at 13 to 14 days to 60 at 23 to 26 days, then dropped to 29 by 30 days of age. Gates and Runner ( 1957) reported the beginnings of follicle responsiveness at 15 days of age, the maximum superovulatory response at 3 weeks, and a decrease in responsiveness thereafter. Ovarian weight has been reported to be most responsive to PMS on day 20 ( Soper, 1963). References to the techniques for induction of superovulation are given in the Bibliography of Techniques.

Sexual maturity normally occurs coincidentally with rising titers of circulating gonadotropin at some time after 4 weeks of age. Precisely when maturity occurs is highly variable and, in addition, such a statement must be interpreted within the framework of the measurement used to determine when "sexual maturity" has been reached. The first observable signs of puberty in females are estrogen-dependent: vaginal introitus and a cornified vaginal smear. Other indices of sexual maturity such as willingness to mate, the ability to conceive and carry a litter to term, and conceivably sexual maturity as measured by the ability to produce weanling-age young have more complex hormonal bases.

The vagina may open as early as day 24 and is often reported open by 4 weeks of age. The first vaginal cornification often occurs 24 to 120 hours after establishment of the vaginal opening, but this is also subject to considerable variability. In addition, estrus, in the sense of willingness to mate, does not always occur on schedule. Mirskaia and Crew ( 1930) reported that mating occurred at the first (vaginal) estrus in 75 and 85 per cent of the cases in two strains; percentages of pregnancy resulting from these first matings, however, were a low 48 and 57 per cent compared to 80 to 90 per cent for more mature animals. The differences inherent in attempts to determine when an animal is fully sexually mature should be obvious.

Attainment of sexual maturity, regardless of the measurement of such development, is a highly variable process. For example, utilizing time of vaginal opening, Parkes ( 1925) stated that his albino mice were usually mature by 7 weeks of age, whereas Engle and Rosasco ( 1927) reported a median age of 35 days and a range of 28 to 49 days also in an albino strain. Vaginal opening in C57BL/6J mice in this laboratory has been observed, in rare instances, as early as 24 days. Two factors contributing heavily to such variation are genetic background and season. Many reports establish that some degree of interstrain variability may occur ( MacDowell and Lord, 1927; Heinecke and Grimm, 1958; Rudali et al., 1957). With respect to seasonal effects, Yoon ( 1955) reported that vaginal introitus occurred at an earlier age in summer than it did during the winter months. He accounted for much of the associated variance by fitting a sine curve to the data, thereby establishing strain differences after accounting for seasonal effects. The effect of season is relatively large. Time of vaginal opening averaged 29 days at the trough of the curve (winter) and 41 days at the peak (summer) for C57BL/10 mice. Vaginal opening, first cornified smear, and first typical estrus are all delayed by experimental exposure to cold ( Barnett and Coleman, 1959).

Considerable work has been done on pituitary-ovarian function in immature mice, much of it directed toward inducing precocious maturity. Smith and Engle ( 1927) inducing precocious maturity. Smith and Engle ( 1927) induced vaginal opening by day 15 after five daily transplants of mature pituitaries. Three daily transplants resulted in ovulation and mating by day 20. Pfeiffer and Hooke (1942) obtained a cornified vaginal smear by day 15, following PMS injection. These studies again indicate the inability of the ovary to respond to pituitary hormones before days 14 or 15. Utilizing estradiol injections, however, Takasugi and Bern ( 1962) were able to induce vaginal opening as early as 5 to 7 days of age, indicating the readiness of the vagina to respond to ovarian hormones at a much earlier age.

A series of papers by Runner, Gates, and Smithberg probably best illustrates induction of (relatively complete) sexual maturity in immature females. Females 30 to 35 days of age were caused to ovulate, mate, and conceive by priming with PMS 60 hours before the desired ovulation time and administering an ovulatory dose of HCG 13 hours before the desired time. Implantation would not occur ( Runner and Gates, 1954), but the eggs developed with normal viability if transplanted to mature recipients ( Gates, 1956). Implantation failure apparently occurred because of incomplete development of corpora lutea, since implantation was accompanied by daily injections of progesterone. Pregnancy was maintained with large doses of progesterone and parturition occurred normally if relaxin was given ( Smithberg and Runner, 1956).

Only a few of the other reports dealing with sexual maturity in mice will be mentioned. Strong and Fuller ( 1958) examined vaginal opening within the framework of selection experiments. Backman ( 1939) utilized a mathematical approach to characterize attainment of sexual maturity. Time of attainment of sexual maturity has been experimentally altered by many factors, including neonatal castration ( Reynaud and Reynaud, 1947) and caloric restriction ( Ball et al., 1947). Histological and histochemical characteristics of developing mouse ovaries and uteri are described by Ben-or ( 1963), Barraclough ( 1955), and Crelin and Levin ( 1955), and in many older works summarized by Snell ( 1941). Little is known about the details of sexual maturation in males. The process, however, is thought to proceed somewhat more slowly than in females ( Parkes, 1925; Bishop and Leathem, 1946, 1948).

GAMETOGENESIS

The mitotic and meiotic phases of gametogenesis show marked chronological differences in females and males. The last of the mitotic divisions of the gonocytes in oogenesis occurs on day 12 or 13 of gestation. The existing cells then enter meiotic prophase and no new cells are formed thereafter. In spermatogenesis, the gonocytes are transformed into spermatogonia during early neonatal life and these continue to divide by mitosis throughout the reproductive life of the male. The discussion of oogenesis, then, is more properly delegated to Chapter 12, while the remainder of this section will describe spermatogenesis. For a complete description of the chromosomal aspects of meiosis, the reader is referred to Chapter 7.

The primordial germ cells, the gonocytes, divide mitotically during prenatal and neonatal life. During early postnatal life, the gonocytes are transformed into spermatogonia. In the mouse, three types of spermatogonia can be recognized: Type A, Intermediate, and Type B ( Oakberg, 1956a). Type A spermatogonia divide four times, usually being transformed in the process into Intermediate type spermatogonia. A few of the resulting cells remain as Type A, enter a period of dormancy of about 207 hours, and then act as stem cells for a new cycle of multiplication ( Oakberg, 1956a; Monesi, 1962). The Intermediate types divide by mitosis again and their progeny become transformed into Type B spermatogonia. The Type B cells undergo mitosis and change into spermatocytes in the preleptotene stage of meiotic prophase. These cells have progressed to the diplotene stage approximately 12.5 days later and then rapidly go through the meiotic divisions. The duration of the complete process for the development of Type A spermatogonia into spermatozoa takes about 34.5 days ( Oakberg, 1956b). The entire sequence of events for spermatogenesis in the mouse, as well as other mammals, has been presented diagrammatically by Roosen-Runge ( 1962).

Not all of the cells survive spermatogenesis. A few spermatogonial cells, particularly Type A, break down in mitosis and thereafter degenerate. A second loss of cells, approximately 13 per cent, occurs in the period from early primary spermatocyte to spermatid. Degeneration has been observed at the first and second meiotic division ( Oakberg, 1956a).

ESTROUS CYCLE

Knowledge of the estrous cycle in the mouse dates from the classic study by Allen ( 1922). Unless interrupted by pregnancy, by pseudopregnancy, or by other events to be discussed later, mice normally tend to display signs of estrus, including mating behavior, every 4 or 5 days. The mouse therefore is a polyestrous mammal. Individual cycles are actually complexes of related secretory, anatomical, and behavioral cycles in which the rhythmic interaction of pituitary and ovarian hormones is fundamental and which, in their totality, have the function of insuring fertilization. The periodicity of estrus observed in mature females is a direct result of cyclic changes that occur in the ovary, which, in turn, reflect altered hypothalamic activity and changes in gonadotropin secretion. The problem of the basic responsibility for such rhythmicity is still not thoroughly understood and relatively little of the research into this problem has utilized the mouse. The key to cyclic reproductive activity apparently lies in the hypothalamus, which communicates with the anterior pituitary by way of a portal system. FSH, a protein, is released by the anterior pituitary and acts to promote follicle growth. Another anterior pituitary protein, LH, aids in the final development of the mature follicle and facilitates production of estrogens by the theca interna cells of the FSH-primed follicle. Further release of LH by the hypophysis results in rupture of the follicle and ovulation. Increasing titers of estrogen during the later phases of follicular growth are thought to act, by way of the hypothalamus, both to suppress further release of FSH and to favor release of more LH. Progesterone, another gonadal steroid, is also produced in the ovary in small quantities during the follicular growth phases. Progesterone, in small doses, promotes ovulation be enhancing LH release. Thus the gonadal hormones produced during follicular growth act on the hypothalamus to suppress further release of FSH while promoting release of LH and hence ovulation. Functional development of the corpus luteum in the mouse is induced by mating and, when this does not occur, gonadal hormone titers decrease allowing succession of a new cycle. The prime factor allowing for the periodicity of estrous phenomena is thought to be cyclic activity in the hypothalamus which is reflected in LH release.

While the basic responsibility of the rhythmicity remains somewhat in doubt, considerable work has been devoted to descriptions of the individual anatomical and secretory cycles which, together, constitute the estrous cycle in mice; these may be discussed in some detail.

Divisions of the estrous cycle

The mouse estrous cycle has been divided into as few as four phases: diestrus, proestrus, estrus, and metestrus; or as many as 13 ( Thung et al. 1956). The latter system consists of a single stage of diestrus, four of proestrous, two of estrus, and six of metestrus. Since the cycle is continuous and a division into so many stages of the cycle would rarely be used, this section will rely on a five-stage description of the cycle ( Allen, 1922). The first two stages (proestrus and estrus) are anabolic stages during which active growth is in progress in various parts of the genital tract. They culminate in ovulation and, where mating occurs, in fertilization. The third and fourth stages, metestrus-1 and metestrus-2, are catabolic stages characterized by degenerative changes in the genital tract. The last (or first) stages, diestrus, is a period of quiescence or slow growth.

The cycle may be conveniently charted by examining vaginal smears. Cellular characteristics of vaginal smears reflect changes in the structure of the vaginal epithelium which, in turn, are dependent upon estrogen and follow a regular and predictable course during the cycle. Three types of cells are found in vaginal smears: leukocytes, cornified epithelial cells, and nucleated epithelial cells. Relative abundance of the various types of cells during the different stages of the cycle are given in Table 11-1. A number of variations from this basic pattern may occur. As estrus approaches, the smear may contain epithelial cells with dark-staining cytoplasm and karyolytic nuclei. Cells intermediate between cornified and nucleated epithelial cells also occur. It should be noted that the smear marking the termination of estrus is often denoted by the presence of clumps or sheets of cornified cells ( Snell, 1941). A more detailed description of the changes in cell type and stainability in vaginal smears of mice may be found in Thung et al. ( 1956).

Ovary

The mouse ovary reveals a marked cycle of ovum and hormone production but little change in weight (except in pregnancy). Mice are spontaneous ovulators with ovulation normally occurring sometime in estrus. Newly formed (nonfunctional) corpora lutea, which persist for two to four cycles, are present in metestrus-1. Counts of the number of mitoses occurring in the germinal epithelium reveal mitotic activity to be least in diestrus, rising slowly through preovulatory estrus, and are characterized by a sudden high spurt of activity in postovulatory estrus. Much of the sequence of oogenesis takes place in this same short postovulatory spurt of activity ( Bullough, 1942). By proestrus the follicles that will ovulate at estrus are definitely set apart by their larger size; follicles average 380 μ in diameter at the beginning of the estrus cycle and reach a maximum size of about 550 μ immediately before rupturing. Most of this change occurs in the last 48 hours.

Not all the follicles which begin the rapid preovulatory growth are destined to ovulate. In mouse ovaries collected in late diestrus, Nakamura ( 1957) found an average of 8.0 follicles per ovary, which appeared to be maturing normally in readiness for the next ovulation. This number had decreased to an average of 7.3 at proestrus and to 4.0 at estrus. The number of aberrant follicles (e.g., second maturation division complete, gross chromosomal aberrations, etc.) averaged 3.3 in late diestrus, 5.3 in proestrus and had decreased to 1.1 at estrus.

"Free" progestin in the plasma has been measured during the various phases of the cycle by Guttenberg ( 1961). An average of 0.5 progesterone equivalents per milliliter was found at diestrus, 3.5 at proestrus, 4.0 at estrus, 2.1 at metestrus-1 and 0.8 at metestrus-2. Plasma progestin levels, therefore, rise well in advance of corpora lutea formation. Estrogen levels, on the other hand, are apparently highest at proestrus as shown by a variety of indicators (e.g. vaginal alkaline phosphatase studies by Váczy et al., 1955). Uterine and vaginal changes during the estrous cycle are the responsibility of these two ovarian hormones, particularly estrogen.

The effect of the ratio of estrogen to progesterone on the vaginal smear has been examined experimentally by Jones and Atwood ( 1942). An adequate dose of progesterone will convert an estrous smear to diestrus regardless of the amount of estrogen given. A relatively large dose of progesterone is needed to inhibit the vaginal response to estrogen, but only a small amount will change an estrous smear to diestrus. Endocrine ramifications may sometimes be complex, however, as illustrated by the finding that adrenocortical neoplasms are responsible for some estruslike cycling in gonadectomized females ( Christy et al., 1950). Green ( 1957) found no change in ovarian sensitivity to injected PMS during the estrous cycle.

Vagina

There is a tendency for the vaginal orifice to gape and the vulva two swell at proestrus and estrus. These signs are variable, however, and of less precision in detecting estrus than the characteristics of vaginal smears. Snell et al ( 1944) suggested that the condition of the cervix (as determined by observation during Artificial insemination) may be of more value than vaginal smears in predicting estrus. An absence of moisture in the cervix, tension of the cervix muscles, and absence of leakage following Artificial insemination are all indicators of estrus.

The successive stages within the vagina itself are briefly summarized in Table 11-1. In proestrus the epithelium consists of three layers. The outer layer is composed of epithelial cells sometimes more or less filled with mucus and with nuclei showing signs of pyknosis. Below this is the stratum granulosum which, with the approach of estrus, becomes the stratum corneum. Third is the stratum germinativum, some seven cell layers in thickness. During proestrus and early estrus the cells of the outer layer are sloughed, producing the characteristic nucleated cell smear. The degree of delamination is not uniform in all parts of the vagina; prior to the onset of estrus the cornified outer layer may be fully exposed in some regions, not in others. Cells are sloughed from the cornified layer during late proestrus and throughout estrus. The onset of metestrus-1 is characterized by the peeling off of the whole layer, with an accompanying rise in the cornified cell count in the smear. During metestrus-2 there is a rise in the nucleated cell count, indicating that in the last stages of the delamination process some of the superficial layers of the stratum germinativum are included. The superficial layers of the stratum germinativum, meantime, have become heavily infiltrated with leukocytes which also appear abundantly in the smear. As a result of the loss of the superficial layers, the vaginal epithelium at diestrus contains only one layer, the stratum germinativum, some three to seven cell layers in thickness. Active growth begins in the stratum germinativum late in diestrus, and by proestrus a stratum granulosum has formed several cell layers below the surface, thus completing the cycle.

Weight of the vagina, either wet or oven-dried, is lowest at diestrus and greatest at proestrus. A comparison of dry-to-wet weight ratios reveals that the vagina is dehydrated at proestrus and estrus, relative to the other stages of the cycle ( Balmain et al., 1956). Several studies have examined biochemical correlates of the process of keratinization in the vaginal epithelium: glycogen content ( Balmain et al., 1956; Biggers, 1953), alkaline phosphatase levels ( Váczy et al., 1955), sulphydryl and disulphide groups ( Asscher and Turner, 1955), and nucleic acids ( Thiery, 1960).

Uterus

The uterus, like the vagina, undergoes a series of anabolic and catabolic changes during the estrus cycle, but they are somewhat less striking ( Table 11-1). In general appearance, the uterus is distended because of the activity of the uterine glands in proestrus and estrus. The distension starts to diminish in late estrus, and in diestrus the uterine wall is collapsed and anemic. The wet and dry uterine weights are lowest at diestrus and heaviest at proestrus; the uterus is relatively hydrated at proestrus and estrus ( Balmain et al., 1956). Glycogen content is greatest at proestrus. The uterine epithelium is composed of low columnar cells in estrus ( Clauberg, 1931). In metestrus-1, degenerative processes become apparent. The basement membrane fades into a pink-staining band which includes the basal sides of the epithelial cells and the superficial stroma. The epithelium loses its definite organization and shows vacuolar degeneration. Leukocytes appear in the region of the basement membrane. In metestrus-2 the degeneration of the epithelium is further advanced, so that almost all the epithelial cells are lost ( Rietschel, 1929). Cell walls at this stage are no longer recognizable and leukocytes are numerous. The uterine glands show minimum activity. The onset of diestrus is marked by the beginning of regenerative processes. Fuxe and Nilsson ( 1963) have examined the ultrastructure of the mouse uterine epithelium and have confirmed that maximum secretory activity occurs at estrus.

Mammary glands

The mammary glands show cyclic growth and regression, though the changes are slight compared with those occurring during pregnancy ( Cole, 1933). In proestrus, buds appear on the ducts, particularly around the periphery of each gland, and large, blunt projections appear on the main ducts near the nipples. In estrus the mammary ducts become dilated, and the buds formed during proestrus elongate. Metestrus-1 introduces regressive changes and by the end of metestrus-2 the ducts are decreased in width and the duct endings collapsed. In diestrus the mammary gland consists of a very open network of narrow, threadlike ducts with comparatively few branches.

Other correlates of the estrous cycle

Slight changes in body weight have been reported for mice at estrus ( Dewar, 1957). Henin ( 1941) described changes in the oviduct correlated with the estrous cycle. Kliman and Salhanick ( 1952) reported a slight relaxation of the pubic symphysis at estrus. Bullough ( 1942, 1946) has described a correlation between mitotic activity in the skin and the estrous cycle.

Time relations of the estrous cycle

Two aspects of the timing of the mouse estrous cycle bear discussion: the occurrence of cycles in relation to lifespan and length of the entire cycle and of its arbitrary divisions. A third aspect of timing, the relationship between the estrous and light-dark cycles, will be discussed in the sections on mating and ovulation.

The onset of estrous cycling has been discussed previously. Several workers have examined the relationship between advanced age and occurrence of cycles. Such a relationship is, apparently, quite variable and dependent upon the particular strain under study. Caschera ( 1959) found increasing irregularity in cycles with advanced age, including some rather prolonged periods of diestrus. Thung et al. ( 1956) also found decreasing numbers of cycles per unit of time in advanced age. Cycles were characterized by somewhat lengthened metestrus and diestrus in older mice, interspersed with some cycles in which full estrus was not obtained, irrespective of breeding history. Strain differences occurred in both the pattern of irregularity which developed with age and in the age of the cessation of cycles, DBA mice showing a prolonged anestrus at the end of life due to ovarian degeneration whereas some other strains continued cycling until death. Boot and Mühlbock ( 1957) reported some ovulation after cessation of fertility. Suntzeff et al. ( 1938) reported that the average depth of the epithelial processes extending into the underlying connective tissue in the mouse vagina increases with age. Jones and Krohn ( 1961) could find no evidence (except in one strain, CBA) that the decline in fertility at older ages was a result of decreased numbers of oocytes. They postulated that such losses in fertility are probably traceable to the hormonal control of the ovary or in the uterine environment.

The average length of individual cycles is usually considered to be 4 or 5 days but this is highly variable and, apparently, easily influenced. Parkes ( 1928, for example, found the following distribution of lengths for 1,000 cycles in unmated albino mice: 2 days, 4 per cent; 3 days; 3 per cent; 4 days, 16 per cent; 5 days, 29 per cent; 6 days, 22 per cent; 7 days, 12 per cent; 8 days, 6 per cent; 9 days, 3 per cent; and 10 to 28 days, 8 per cent. Estrus itself has been variously reported to last from ½ to 9 days ( Allen, 1922; Snell, 1941). Probably, considering the environmental effects which will be discussed shortly, the best set of data relative to length of the various phases of the "average" cycle comes from Boot (in Van Ebbenhorst Tengergen, 1955): early proestrus, 11 hours; late proestrus, 21.4 hours; estrus, 20.7 hours; metestrus, 21.8 hours; and diestrus, 21.8 hours for a total of 104 hours or 4.3 days.

The causes of such variation in the length of the cycle can be, initially, divided into genetic and environmental (realizing that these are not necessarily independent). Strain differences in cycle characteristics have been the subject of numerous studies which will not be cited here. In general, it may be said that various strains do differ in length of the cycle and, sometimes, of its phases. In addition, some of this type of variation has been traced to different thresholds for the vaginal response to estrogen ( Mühlbock, 1947). Worthy of note, with respect to genetics and estrous cycles, is a paper by Mizuhara et al. ( 1958), who developed a graphical method for more accurate comparison of estrous cycles between strains.

Environmental variation may be classified into that stemming from the social and that from the nonsocial environment. Noteworthy in the second category are effects of such factors as season and diet. Seasonal effects and their interaction with genetic background have been established. Mice of strains A and C57 averaged cycles of 5.2 and 5.4 days in length in February, whereas both averaged 5.8 days in May. The longer cycles in May were due to a somewhat prolonged proestrus and, particularly, to a much longer metestrus. Strain A mice were characterized by a longer estrus and a shorter diestrus with metestrus and proestrus about the same length as occurred in C57 females ( Laguchev, 1959). The relationship between diet and estrous characteristic has been examined many times (e.g., Hallberg and Visscher, 1952; Lugo, 1959). Worthy of note are the effects of estrogenic plant substances (e.g., Cranston, 1945; East, 1955).

A series of findings implicating the social environment as a prime source of variation in estrous cycles casts doubt on the validity of many earlier reports on cycle characteristics and, actually, poses the problem: What is a "normal" estrous cycle? Estrus is somewhat suppressed (due to pseudopregnancy or prolonged diestrum) in mice crowded in all-female groups ( van der Lee and Boot, 1956; Whitten, 1959; Marsden and Brosom, 1965). This phenomenon has, in part, been traced to olfactory-mediated stimuli. In addition to the effects of the all-female environment, olfactory stimuli originating from a male are known to, in a sense, override the effects of the female stimuli by regularizing some of the abnormal cycles found under crowded conditions ( Whitten, 1957). The effects of a male (i.e., to regularize and actually accelerate estrus) have been mimicked utilizing male urine alone ( Marsden and Bronson, 1964). The relationship between olfactory stimuli and mammalian reproduction has been reviewed by Parkes and Bruce ( 1961). In addition to olfactory stimuli, neurotropic stimuli, loosely characterized as "social stress" and stemming from the increase in behavioral competition accompanying crowding, affect estrous characteristics. Crowding, particularly when accompanied by some degree of social competition between males, is known to alter some aspects of central nervous system activity and increase synthesis and release of ACTH ( Christian, 1960) and it is known that increased ACTH production may, directly or indirectly, alter ovarian function ( Christian, 1964).

Postpartum estrus

The interval between parturition and the following ovulation in mice has been reported to be about 14 to 28 hours ( Long and Mark, 1911). Runner and Ladman ( 1950) have examined this relationship more closely in light of the fact that they found parturition occurring at random across a 24-hour period whereas, as will be shown in following sections, ovulation and mating are strongly influenced by the light cycle. Ovulation during the postpartum estrus is, apparently, not timed exclusively by either the light cycle or the time of parturition. They found good predictability in ovulation times only when both factors were considered, the tendency to ovulate nocturnally being incompletely countered by a tendency to ovulate 12 to 18 hours after parturition. Cornification of the vagina is not complete at postpartum estrus, and the cornified cell content of the smear normally never reaches 100 per cent. Fertile matings are less often obtained during this period than during the course of the normal cycle. In addition, there is less fluid in the uterus than during a normal estrus ( Merton, 1939).

OVULATION

Ovulation occurs spontaneously during estrus in mice whether or not mating has occurred. However, ovulation may not occur at every estrus, particularly in young virgin females ( Togari, 1927), and estrus may not always accompany ovulation ( Young, 1941). It should be remembered that the two phenomena, estrus and ovulation, have different endocrinological bases, estrus being dependent upon gonadal hormones whereas ovulation occurs in response to gonadotropins. Following initial stimulation of follicular growth by FSH, LH titers begin to rise, resulting in drastically increased secretion of follicular liquor and, finally, ovulation. Experimental production of ovulation by injections of PMS and HCG is discussed in the section on sexual maturity.

Strain differences in the state of ovarian development reached at birth are known ( Jones and Krohn, 1961). However, by 3 to 6 days post partum the oocytes usually have a follicular epithelium ( Brambell, 1927). The primordial follicles in the adult consist of a single thin layer of cells surrounding the germ cell and lie near the ovarian epithelium. The oocytes measure approximately 13 μ in diameter, and the entire follicle about 16.5 μ ( Brambell, 1928). As the oocyte and follicle increase in size they gradually move towards more central regions of the ovary. As the egg and follicle approach maturity they, in turn, resume their peripheral position on the ovary.

The epithelial cells of the follicle divide and transform into cuboidal and thence columnar cells as the oocyte begins to enlarge. When the egg has reached maximum growth, the multilayered follicular epithelium develops a fluid-filled cavity, the antrum. In the mouse, the growth of the follicle relative to the growth of the oocyte can be divided into two phases. During the first phase both the oocyte and the follicle grow rapidly, in a correlated fashion. At the end of this phase the oocyte measures 70 μ in diameter as compared to 125 μ for the follicle. During the second phase, there is no significant increase in the size of the oocyte, but the diameter of the follicle increases to about 55o μ (under the influence of LH) just before ovulation. The follicular growth during the second phase is due largely to distention of the antrum with fluid.

Just prior to ovulation the egg undergoes the final stages of maturation. The centrosome divides into two centrioles, the asters are formed, and the rest of the spindle apparatus appears. In the mouse the spindle lies tangential to the surface ( Long and Mark, 1911). The first meiotic division is completed and the first polar body is cut off and comes to lie beneath the zona pellicida. The second meiotic division begins in the oocyte nucleus but does not proceed beyond metaphase. The nucleus will remain in this condition, normally, until the sperm has entered the egg cytoplasm ( Snell et al., 1940).

The period of estrus and the time of ovulation are normally controlled by the diurnal rhythm of light and darkness. Reversing the time of light and darkness reverses the time of estrus and ovulation ( Snell et al., 1940; Snell et al., 1944; Braden, 1957). Published accounts differ considerably as to the time of ovulation in relation to the onset of estrus. Ovulation has been reported to occur at or near the beginning of estrus ( Brambell and Parkes, 1927; Lewis and Wright, 1935; Snell et al., 1940), and at or near the end of estrus ( Allen, 1922; Togari, 1927). In MacDowell-Bagg albino mice, ovulation usually took place between midnight and 3:00 AM, while mating most commonly occurred between 10:00 PM and 1:00 AM. Thus, in this strain, mating preceded ovulation by an average of only 2 hours. The interval varied considerably, ranging from 1 to 3.75 hours ( Snell et al., 1940). Ovulation within 15 minutes of mating has been noted ( Lewis and Wright, 1935).

Investigations in which the light-dark cycles were Artificially controlled have supported the conclusion that the midpoint of the ovulation period is determined more by the midpoint of the ovulation period is determined more by the midpoint of the dark phase than by its onset or completion. Averaging the results of three stocks, Braden ( 1957) found the midpoint of the ovulation period to occur around 3.5 hours after the midpoint of the dark period when animals were kept on a 10-hours-dark to 14-hours-light cycle. The delay averaged 4.75 hours after the midpoint of the dark period in animals on a 4-hours-dark to 2-hours-light cycle. For two inbred strains, 129/Rr and BALB/cGn, the midpoint of ovulation was 4.5 to 5.5 hours after the midpoint of the dark period in a 7-hours-dark to 17-hours-light cycle ( Whitten and Dagg, 1961).

Some of the apparent discrepancies in earlier reports may have been due to using mating time rather than the light-dark cycle as a reference point. Undoubtedly, some of the reported differences are due to interstrain variation. Three noninbred stocks (L, C, and PCT) were found to differ in the midpoint of the ovulation period, indicating that mechanisms controlling the time of ovulation are modified according to the genetic constitution of the stock ( Braden, 1957). Braden also noted that the interval between mating and ovulation was approximately 5 hours, somewhat longer than the 2-hour interval found by Snell et al. ( 1940). The time required for ovulation of 75 per cent of the eggs released by any one mouse has been estimated as ½ to 1 hour ( Branden, 1957).

MATING

Pertinent literature on sex behavior, including some description, is covered in Chapter 33. Mating in house mice is normally detected by the occurrence of vaginal plugs. Such plugs are formed by a mixture of the secretions of the vesicular and coagulating glands of the male and usually fill the vagina from cervix to vulva. Occasionally, smaller, less conspicuous plugs are formed, a consideration more common in postpartum matings. Plugs persist for 16 to 24 hours and may last as long as 48 hours ( Parkes, 1926).

The efficiency of utilizing plugs to predict pregnancy is usually high. For example, Snell ( 1941) reported that, among two strains of mice, 80 to 90 per cent of the mature females showing a plug became pregnant. However, like so many biological parameters in laboratory mice, predictability of pregnancy from vaginal plugs is strain-dependent. Fainstat ( 1951) reported that only 14 per cent of his strain A females with plugs became pregnant, whereas comparative figures ranged from 29 to 100 per cent in four other strains. Housing conditions during the postinsemination period were not described. Conceivably this could account for some of the large differences, but that strains differ in this respect is certainly true. Eaton ( 1941) suggests that the proportion of successful matings of hybrids might be a factor amenable to genetic manipulation.

Receptivity among laboratory mice is not limited to estrus but may take place during proestrus or metestrus-1 (as indicated by vaginal smears). Fowler and Edwards ( 1957) reported good success in predicting whether or not mating would occur by the examination of a single vaginal smear and, in addition, presented the following frequency distribution of inseminations according to the type of vaginal smear observed in conjunction with the plugs: proestrus or early estrus (moderate numbers of nucleated epithelial cells, few or no leukocytes), 57 per cent (of 7); estrus (moderate numbers of cornified and some nucleated epithelial cells), 83 per cent (of 12); late estrus (moderate numbers of cornified cells), 36 per cent (of 11); metestrus-1 (many clumped cornified cells), 22 per cent (of 9); and none (of 9 and 22, respectively) in metestrus-2 or diestrus.

The length of time between pairing and insemination is strongly dependent upon housing conditions of the female prior to pairing. Whitten ( 1956a, 1957) first described the phenomenon of postpairing synchrony of estrus among females previously housed together. Such synchrony manifests itself as a nonrandom frequency distribution of inseminations (with respect to days) during a 4- to 5-day postpairing period. Specifically he found a third-day peak in inseminations with correspondingly lower levels on days 1, 2, and 4. Exteroceptive factors arising both from the all-female grouping prior to pairing and from the male at the time of pairing contribute to such synchrony ( Marsden and Bronson, 1965). As mentioned previously, stimuli which may or may not be olfactory-mediated tend to suppress the estrous cycle among crowded females. Release from such stimuli by removing the female for individual pairing with a male, as well as olfactory stimuli associated with male urine, acts to recommence her cycle and leads to most females attaining estrus on the third day after pairing.

Matings may occur out of phase with the light-dark cycle on the first day after pairing depending upon the time of pairing. Although it is normal for behavioral estrus, mating, and ovulation all to take place somewhere during the dark cycle, mating may take place throughout much of the light cycle if paring with a male is done just after the light phase begins. Such out-of-phase mating is probably restricted to animals that would normally have mated during the preceding dark phase. For example, in a recent series of C57BL/6J females paired with a male at 9:00 AM (light phase 5:00 AM to 7:00 PM) in this laboratory, 51 per cent of 99 plugs formed during the first 24 hours after pairing were formed before 4:00 PM. The corresponding figures for the second and third days after pairing were, respectively, 6 per cent of 54 and 4 per cent of 50. It is probable that only insemination itself is out of phase in these cases and that some of these matings would be infertile.

The interactions between the environment, central nervous system activity, and the gonadal hormones in producing behavioral symptoms of estrus (spontaneous activity, sexual receptivity) in females or mating behavior in males cannot be denied. Unfortunately the mouse, as opposed to such mammals as the rat and guinea pig, has been little utilized in studies designed to elucidate the physiological causes of mating behavior ( Young, 1961). Engel ( 1942) reported somewhat ambivalent sex-behavior changes in males after sex-steroid injections. Induction of estrus and mating in immature females has been accomplished as described in a previous section on sexual maturity. Several early investigators reported induction of mating by estrogen treatment alone, e.g., Allen and Doisy ( 1923). Ring ( 1944) reexamined this problem and found that, regardless of dose, not all females treated with estrogen would mate. When both progesterone and estrogen were given, 100 per cent of the females mated. Fowler and Edwards ( 1957) injected 1 IU of PMS followed in 40 hours by 2 IU of HCG or 3 IU of PMS followed by 3 IU of HCG into mature mice selected at random with respect to estrus and found that mating occurred in 75 per cent and ovulation in 99 per cent of the animals within 20 hours. Three-fourths of the injected animals became pregnant as opposed to 90 per cent for controls. Partial dependence of dose requirements of PMS and HCG on genetic background has been demonstrated ( Edwards et al., 1963).

Artificial insemination procedures, consisting of insemination via a hypodermic needle inserted through the cervix, followed by copulation with a castrated male have been described by Snell et al. ( 1944). This procedure yielded, in general, 70 per cent of fewer pregnancies. Kile ( 1951) injected sperm through the uterine wall 1.5 to 4.5 hours after copulation with a vasectomized male and obtained 72 per cent fertility.

Many cases of mating at various times during pregnancy have been reported; for example, Mirskaia and Crew ( 1930), Bilewicz and Mikiewiczowa ( 1954), and Bloch ( 1958).

FERTILIZATION

Fertilization usually occurs in the ampulla or the upper end of the oviduct, and sperm may be present in this area for several hours before ovulation occurs. Although the main mass never reach the ovarian end of the oviduct, travel may be rapid by those that do attain this distance (as little as 15 minutes after ejaculation; Lewis and Wright, 1935). A relatively small number of sperm (fewer than 100) have been found near the eggs at the time of fertilization ( Braden and Austin, 1954a).

The mechanisms for transport of sperm through the uterus and Fallopian tubes are not completely understood. Motility of the sperm appears to play only a small part; contractions of the uterus and possibly the oviducts probably hasten transport ( Parkes, 1960). Sperm that reach the Fallopian tubes retain their fertilizing ability for about 6 hours, even though their motility has been reported to persist for 13.5 hours ( Merton, 1939). Sperm remaining in the uterus are phagocytosed by polymorphonuclear leukocytes within 20 hours after mating ( Austin, 1957). The egg has usually completed the first maturation division by the time ovulation occurs. The first polar body is present and the second maturation spindle has formed. If the egg has not yet completed the first maturation division it does so very quickly after ovulation. The polar body generates as early as the one-cell stage or as late as the morula ( Lewis and Wright, 1935). Each egg is surrounded by a zona pellucida and, external to this, a mass of cumulus cells, the cumulus oophorus. The cumulus cells are adhesive; consequently, all the eggs from one ovary are frequently gathered into a clump by attachment of the masses of cumulus cells. Sperm penetrate the cumulus cells and reach the outer egg membrane in about 2 hours ( Lewis and Wright, 1935).

More than one spermatozoon can enter the perivitelline space. However, usually only one sperm penetrates the vitellus and fertilizes the egg. As the egg ages, its capacity to prevent polyspermy diminishes. In one study the local application of heat to the Fallopian tube raised the frequency of polyspermy from 0.3 to 3.8 per cent, but delayed coitus did not alter the frequency ( Braden and Austin, 1954c). As many as four supplementary sperm have been observed in the perivitelline space. The frequency distribution of eggs with extra sperm differed from the expected distribution, indicated that fewer sperm penetrated into the zona pellucida than expected by chance. The entry of the first or fertilizing sperm seems to restrict the admission of additional sperm into the zona pellucida ( Braden et al., 1954).

The egg of the mature mouse is capable of being fertilized and producing normal embryos for about 10 to 12 hours after ovulation ( Long, 1912; Braden and Austin, 1954b). Eggs undergo a gradual aging in which functions successively disappear. An early manifestation is the production of abnormal or inviable embryos. Further deterioration leads to abnormalities of fertilization and syngamy and even to loss of protection against polyspermy. Unfertilized eggs undergo degeneration in the Fallopian tubes and uterus. Frequently these cells fragment, producing clusters of two or more cells resembling normal division stages. Closer examination, however, reveals that cell sizes are usually atypically unequal and the nuclear material is not distributed normally. Some cells resulting from the spontaneous fragmentation may be without a nucleus, whereas others appear to have more than one. See Chapter 12 for additional comments on fertilization.

GESTATION

While estrogens assume primary (but not complete) responsibility during the estrous cycle, progestagens may be thought of as assuming a primary role for gestational success. Neither of these statements is of course completely true since progesterone, among other things, is required for successful mating and plays a prime role in ovulation, while it is known that estrogens act synergistically with progestagens to insure a successful pregnancy.

Functional corpora lutea are not found in mice unless mating (or cervical stimulation) has occurred. The act of mating produces widespread activation of the nervous system including the hypothalamus, thereby promoting release of luteotrophic hormone (LTH) by the anterior pituitary. LH administration can cause luteinization in the mouse ovary even without ovulation, but the development of a functional corpus luteum is under the control of LTH (prolactin in mice).

There is probably a neural block to LTH release which prevents functional corpus luteum formation and, consequently, high titers of progesterone during infertile estrous cycles. If mating occurs, functional corpora lutea develop, the uterus comes under progestational dominance, and ovulation and succeeding estrous cycles are blocked. Implantation normally occurs about 5 days after insemination coincident with rising progesterone levels ( Forbes and Hooker, 1957). Placental hormone secretion is apparently of less importance for the maintenance of pregnancy in mice than it is in many other species, since gonadectomy at any time during gestation is followed by termination of pregnancy.

Sterile matings or cervical stimulation with a glass rod may induce pseudopregnancy. The average interval between a sterile mating and the next estrus in mice has been reported to be about 11 days ( Deanesly, 1930), but later reports have shown this to be quite variable. The initial stages of pseudopregnancy and pregnancy are similar in hormone responses ( Atkinson and Hooker, 1945). Following a sterile mating the corpora lutea become functional, sensitizing the uterine epithelium which can then respond to trauma. In the pseudopregnant mouse, the maximum capacity for deciduoma formation following local injury of the uterus occurs about 3 days after mating. By 5 days after mating the sensitivity is almost lost ( Parkes, 1929). Contrary to findings in some other mammals, electrical stimulation is relatively ineffective as a means of producing pseudopregnancy in mice ( Shelesnyak and Davies, 1953).

Gestation in the nonsuckled mouse normally lasts 19 to 21 days. Fekete ( 1954) has calculated that a female bearing a litter of 5 or 10 gains, respectively, 2.5 or 3.5 times as much weight during pregnancy as a female with a litter of one. Length of gestation, like most other aspects of mouse reproduction is strain-dependent. For example, in both DBA and C57BL strains, the modal length of gestation has been reported to be 20 days, but a large proportion of C57BL than DBA females delivered at 19 days and, in contrast, a larger proportion of DBA than C57BL females gave birth on the 21st day ( Fekete, 1941). Hybrid mice may have shorter gestation periods than inbred mice. Superfetation has been reported for the mouse ( Stowell, 1941); however, as in most other mammals, this is apparently a rare phenomenon. Superfetation has been induced experimentally in mice ( Edwards and Fowler, 1958).

Time of parturition is probably subject to both genetic and environmental influences. Runner and Ladman ( 1950), examining the relationship between the light-dark cycle and time of parturition, reported that in no 4-hour period did fewer than 12 per cent or more than 20 per cent of the births occur. In an older study using a different stock of mice it was found that two-thirds of the births occurred between 4:00 PM and 4:00 AM (dark phase) ( Merton, 1938).

Gestation and lactation often proceed simultaneously but under these conditions the gestation period is lengthened because of a delay in implantation. If only one or two young are being suckled, the prolongation of the gestation period does not exceed 7 days. With three or more young suckling, prolongations up to 12 or 13 days are not uncommon and may go as high as 16 days ( Brambell, 1937; Fekete, 1940).

The reproductive potential of mice is high. Some females in some strains may consistently ovulate 10 or more ova and the reproductive lifespan of some strains approaches 2 years. When one considers the relatively high number of ova that may be shed, the fact that pregnancy and lactation may occur simultaneously, and the fact that some strains may reproduce until quite late in life, the total production of young which could occur is impressive. For many reasons such production is almost never realized. Some females never produce young, others show inconsistent production, and, even among those reproducing more or less consistently, there is normally some degree of loss between ovulation and parturition.

The number of ova shed, as indicated by corpus luteum counts, is usually lower for the first pregnancy but remains fairly constant thereafter. It is normal, however, to expect litter size to decrease in older age because of an increasing level of postovulation loss of ova or intrauterine problems ( Hollander and Strong, 1950) and reproduction usually ceases entirely, or at least becomes very sporadic, in older animals. The usual pattern in litter size is a rise for the first few litters followed by a gradual decline until sterility or death. The number of young born in any given litter is usually between 1 and 10 but is too variable, due to genetic and environmental influences, to justify speaking about "an average litter size" of mice.

The degree of embryonic loss is highest in the earliest stages of pregnancy. Hollander and Strong ( 1950) found an average level of postimplantation mortality of 15 per cent in their stocks. Mortality was noted in all stages of embryonic life but 72 per cent of the observed loss occurred in the first 3 days after implantation. Loss of ova before implantation would further decrease the realized reproduction. One factor not correlated with intrauterine loss was crowding of embryos in the uterus. Bowman and Roberts ( 1958) also reported little correlation between the number of implanted blastocysts and the amount of intrauterine mortality but did not find that the loss of eggs before implantation was correlated with the number shed.

The possible causes for decreases in the reproductive potential are many and may, initially, be categorized as genetic or environmental. Most laboratory mice are inbred to some extent and inbreeding is known often to be accompanied by some degree of reproductive inefficiency. For example, a decrease of 0.56 young per litter for each 10 per cent increase in the inbreeding coefficient was reported by Bowman and Falconer ( 1960). Starting with 20 lines of mice, only three lines were surviving by the time the inbreeding coefficient had reached 76 per cent. The causes for losses of reproductive efficiency which accompany selection or inbreeding experiments are probably manifold. Examples are: increased preimplantation loss of eggs ( Falconer and Roberts, 1960), low libido of the male, or hypofunctioning of the adenohypophysis of the female ( Fowler and Edwards, 1960).

The increased demands placed on a female during pregnancy make her susceptible to many environmental influences. Biggers et al. ( 1958) have reported increased pre- or postimplantational mortality in environments that were either too hot or too cold. Barnett ( 1962) found that mice maintained in a cold environment generally began breeding later with a longer time period between litters and, as a result, produced about half as many young as controls. Mice of one strain, however, produced about the same number of young as controls because of a twofold increase in their reproductive life span. As another example of the possible interactions between genetic background and the environment in producing reproductive inefficiency, Hoag and Dickie ( 1962) reported that strains may differ with respect to the optimum level of protein and fat required for most successful reproduction ( Chapter 5). An example of a common environmental stress placed on pregnant females in the laboratory is the report by Runner ( 1959) who found that gestational success in 129/J mice was decreased by daily handling. Experimentally Weir and DeFries ( 1963) have demonstrated that enforced swimming and activity decrease the number of births.

Pregnancy and pseudopregnancy, like some other aspects of mouse reproduction, are susceptible to alteration by the social environment. Olfactory stimuli are known to induce true pseudopregnancy (as shown by deciduoma formation) in crowded all-female groups (van der Lee and Boot, 1955, 1956). Biancifiori and Caschera ( 1963) reported that 46 per cent of the 35 cycles examined in females which had mated with a vasectomized male were pseudopregnancies. Comparable figures for unmated females housed five per cage and anosmic females maintained one per cage were, respectively, 14 per cent of 150 and none of 54. In addition, a series of papers by Bruce has disclosed that olfactory stimuli originating from a strange male may prevent implantation of the blastocysts or pseudopregnancy. Following exposure to a strange male the female returns to an estrous condition within 7 days after insemination. Any male other than the particular stud male will accomplish this and, if the strange male is of a different strain than the stud male, the proportion of females showing blocked pregnancies is increased still further ( Bruce, 1960; Bruce and Parrott, 1960; Parkes and Bruce, 1961). Experimental evidence suggests that the mechanism involved in this phenomenon includes a failure of the normal luteotrophic activity of the pituitary ( Parkes, 1961). This phenomenon may not occur in many of the highly inbred strains. Testing of adequate numbers of C57BL/6J, SWR/J, SJL/J, and CBA/J strains at The Jackson Laboratory revealed no effect of postinsemination exposure to a nonstud male.

The role in olfaction in mouse reproduction may be more important than previously thought. An intimate relationship has been demonstrated by the large degree of inhibition imposed on reproduction by ablation of the olfactory lobes ( Whitten, 1956b; Lamond, 1958). This may be compared to a study by Chase ( 1941), who found normal development of sexual maturity in a stock of mice suffering from congenital eyelessness.

Stimuli, other than olfactory, originating in the social environment are also known to affect gestational success. Crowding of mice, particularly when accompanied by some degree of fighting between males, is known to have profound effects on several endocrine systems. That such a situation is accompanied by increased pre- and postimplantational mortality is well documented ( Christian and LeMunyan, 1958). Bruce ( 1954) has confirmed that monogamous pairs show the best production but, considering the savings of space and numbers of mice, has concluded that one male and two females per cage is the most efficient housing method with respect to maximum production of mice. There can be little doubt that the relationship between the number of mice per cage on the one hand and genetic and (nonsocial) environmental factors on the other is complex and highly variable.

LACTATION

The suckling period is a critical period, in terms of development, in the life of the mouse. It has been estimated that maternal influence accounts for about 72 per cent of the variance associated with body weights taken at 12 days of age ( Cox et al., 1959. The normal (average) duration of lactation in mice is about 4 weeks but this is somewhat variable undoubtedly, at least in part, because of strain differences. Milk production is not constant throughout lactation. Production rises for about 10 days and then declines until weaning.

Nandi ( 1959) has presented extensive work on the hormonal control of mammogenesis and lactogenesis and has included a literature review. A series of three papers by Munford ( 1963) deals extensively with histological and biochemical characteristics of mammary glands during pregnancy, lactation, and involution.

SUMMARY

Characteristics of the process of reproduction in laboratory mice, while different in detail, bear general similarity to those found in many other mammals. As an attempt to classify their reproduction, mice may be said to be polyestrous; they are spontaneous ovulators in which the formation of luteal tissue is induced by mating; and, in addition, like many other rodents, they possess a high reproductive potential. For a variety of reasons such a high capacity for production of young is almost never fully realized. One characteristic encountered time and again while reviewing the literature for this chapter is the variability found among reports dealing with reproduction in "the mouse"; so much that difficulty is encountered when one attempts to discuss normal parameters of reproduction. Strain differences have been found in almost every instance where they have been sought and, in addition, most aspects of mouse reproduction are amenable to alteration by one or many environmental factors. These range from physical aspects such as light or temperature to the presence or absence of other animals or even their odors. An important category of causes of variability resides in the interaction between genetic background and environment. Strains differ in their reproductive responses to the environment; for example, in their optimum dietary needs or in their capacity to respond to their social environment. Because of such variability it should be emphasized that all exact measurements given in this chapter (e.g., time of ovulation with respect to the light-dark cycle) should be suspect unless the same stocks of animals are used and the same environmental conditions prevail.

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