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4.3.1 Puberty

The onset of puberty — when ovulation first occurs in a female, and when males have achieved full spermatogenic activity — is variable even among different animals within the same inbred strain. Although it is possible for some outbred females to reach puberty by the age of 4 weeks, the majority of females from most inbred strains first ovulate naturally between 6 and 8 weeks after birth (Table 4.1). Numerous environmental factors appear to have an effect on the timing of this event (Whittingham and Wood, 1983). Exposure to adult males or their urine can bring it on sooner, whereas adult females or their urine may retard its onset. Furthermore, 3-6-week-old females can be induced to ovulate with a specific regimen of hormone treatment as described in Section The onset of male puberty in most laboratory strains usually occurs between 34 and 38 days, however, it is sometimes possible for non-inbred males to reach sexual maturity by 30-32 days after birth. Thus, if one does not want littermates to mate with each other, they should be separated according to sex before the appropriate age is reached.

4.3.2 The estrus cycle

The normal estrus cycle of a laboratory mouse is 4-6 days in length. The cycle has been divided into four phases which are distinguished by changes in physiology, morphology, and behavior. (1) The proestrus portion of the cycle begins when a new batch of eggs reach maturity within ovarian follicles that are ripe and large. External examination of the female will usually show a bloated vulva with an open vagina. (2) Estrus begins with the ovulation of fully mature oocytes. The vulva remains in an extended state with an open vagina, and females are maximally receptive to male advances. When mice are maintained on a standard light-dark cycle, the estrus phase will usually begin soon after midnight and last for 6-8 hours. (3) The metestrus phase follows, when mature eggs move through the oviducts and into the uterus. The vulva is no longer bloated, and the vagina is now closed.

At the end of metestrus, a physiological branch point occurs with the direction to be taken dependent on whether a successful copulation has occurred. The act of successful copulation induces hormonal changes that prepare the uterus for a pregnancy which will ensue under normal circumstances. However, a sterile copulation — one that does not lead to fertilization — can induce a state of pseudopregnancy (see Section 6.2.3). A pseudopregnancy can extend the metestrus phase by as long as 10-13 days.

(4) If pregnancy does not occur, the metestrus phase is ultimately followed by the last phase of the estrus cycle, diestrus. Unfertilized eggs are eliminated, the vagina and vulva are at a minimum size, and new follicles begin to undergo a rapid growth for the next ovulation. (The proestrus and estrus phases together constitute the follicular phase; the metestrus and diestrus phases together constitute the luteal phase.)

4.3.3 Mating

Once animals have been together for more than a few days, mating will be restricted to the late proestrus/early estrus portion of the female cycle. It is only during this period that a female will be receptive and that a male will normally be interested. (However, in some instances, when a new couple is first brought together in a cage, the male will mate with his partner, irrespective of her estrus phase). Mating typically occurs over a period of 15-60 minutes with clear strain-specific differences: DBA males are quick (20 minutes) and BALB/c males are slow (one hour) according to Wimer and Fuller (1966). The male first examines the female genitalia and then mounts his mate and withdraws from one to one hundred times until ejaculation occurs during a final mounting. The male is quiet for a short period of time and then resumes normal activity. Although a full sperm count is not built up again for two days, it is possible for a male, especially an outbred one, to mate with up to three females in a single night, causing all to become pregnant. Different inbred strains have very different average times for recovery of libido, defined operationally as the time between attempted matings. DBA/2 mice can mate again within 1 hour, whereas B6 males usually wait for 4 days (Wimer and Fuller, 1966).

In some instances, one may want to maximize the rapid output of offspring from a single male. This situation could arise with rare genotypes such as new mutants or first generation transgenic founders. For this purpose, a single male can be rotated among sets of females (two or three per cage) in three or four cages. The factors that play a role in the length of each rotation have just been discussed: the length of the estrus cycle, the time it takes for a male to recover a full sperm count, and the libido recovery time. Together, these factors suggest an optimal rotation period of 4 days in each cage. For full optimization of offspring output, a male should receive two new, 8-week-old, virgin females in his cage, every 4 days.

4.3.4 Fertilization

Fertilization takes place in the upper reaches of the oviduct (a region referred to as the ampulla). The egg remains viable for 10-15 hours after ovulation, although a gradual aging process slowly reduces the probability that fertilization will occur. Fertilization causes an immediate activation of the egg and induces the completion of the second meiotic division, which leads to the formation of the second polar body within two hours.

The actual process of fertilization can be divided into a series of highly ordered steps that lead ultimately to the joining of a single sperm cell with an ovulated egg (Wassarman, 1993). The first step in this process occurs with the binding of multiple spermatozoa to the zona pellucida, a thick extracellular coat that surrounds the egg. The association between the zona and the sperm surface triggers the acrosome reaction which affects an elongated sperm-specific membrane-bound organelle just below the surface that contains a specialized protease called acrosin. The acrosome reaction is a form of exocytosis that results in the complete loss of the plasma membrane overlying the acrosome in hybrid vesicles along with the outer acrosomal membrane. The acrosomal contents are released, and these allow the resulting "acrosome-reacted" sperm to use protease to digest its way through the zona pellucida to reach the perivitelline space between the zona and the egg plasma membrane. Finally, fusion occurs between the egg plasma membrane and the plasma membrane overlying the equatorial region of a single sperm cell. Fusion leads to the activation of the egg and the initiation of embryonic development.

The ultimate fusion reaction is not species-specific and can occur between heterologous gametes when the zona pellucida is first removed from the egg. Thus, in general, the main biochemical barrier to cross-species fertilization appears to lie within the initial interaction between the sperm plasma membrane and the egg zona pellucida. The specificity of this interaction implicates the existence of specific complementary molecules on egg and sperm, referred to respectively as the "sperm receptor" and the "egg binding protein" or EBP. The sperm receptor has been identified as a specific zona protein called ZP3 (Wassarman, 1990). The identity of the sperm surface EBP is still under investigation with multiple candidates described to date.

4.3.5 Determination of copulation and pregnancy

After a successful copulation has been completed, particular components of the male ejaculate will coagulate to form a hard plug that occludes the entrance to the vagina. The plug is a coagulum of fluids derived from both the vesicular and coagulating glands, and as such, it can be produced even by a vasectomized male. Usually the plug is visible through a simple visual examination of the vulva. In some instances, a probe will be required to detect a plug located further back in the vagina. The most common probe used for this purpose is a simple dental tool with a blunt end. "Plugging" should be performed as early as possible in the morning after a potential mating. By noon, some inbred strain plugs will begin to disappear, however, most will persist for 16-24 hours after copulation. Plugs formed by outbred mice can persist for several days.

Later in the pregnancy, from 10-12 days postconception and beyond, it becomes possible to feel the maturing fetuses within the uterus by simple palpation. Pregnancy palpation is most readily carried out on older, multiparous females who have looser skin and are more accustomed to being handled. Right-handed workers should hold the female in the left hand (left-handers should hold the mouse in the right hand), with the thumb and forefinger grasping the skin behind the neck, and the smallest finger holding back the tail. The other fingers of this hand should be brought in behind the mouse to arch her forward. When the female is securely held by one hand and relatively calm, one should use the other hand to close down firmly on the abdomen close to the spine on one side at a time with the forefinger and thumb and then gently move the fingers out. Initially, a pregnant female will seem to have a string of beads on each side of her body. As development proceeds, these "beads" will mature into larger, more defined shapes. With experience, this method can be used to determine the gestational stage of a pregnancy to within a single day.

It is possible to identify a state of pregnancy in young females by a simple visual inspection that does not even require one to handle the animal. The gestational day at which this becomes possible is greatly dependent on a number of factors including the age of the female, the number of fetuses inside, and whether she has given birth previously. For first-time pregnant females carrying large litters, tell-tale bulges from the center of her body can be detected by day 15. At the opposite extreme, older multiparous females with small litters never "show" in this way. Fortunately, these older animals are easier to palpate when a prenatal determination is required.

4.3.6 The gestational period

The gestation period for the mouse ranges from 18 to 22 days. Different strains have different averages within this range but even within a single strain, and even for a single female, there can be significant differences from one pregnancy to the next. Many different factors can have an effect on the length of pregnancy. For example, larger litters tend to be born earlier (Rugh, 1968), as is the case with humans as well. Non-inbred females tend to have shorter pregnancies then inbred ones, but this may be simply because they tend to produce larger litters as well as larger pups. Birth occurs most frequently between the hours of midnight and 4:00 A.M. when animals are maintained under a standard light-dark cycle; however, it can occur anytime of the day or night.

The gestation period can be greatly extended when the pregnant mother continues to nurse a previous litter. Prolongation up to 7 days is not uncommon, and birth can sometimes be pushed back by as many as 16 days (Grüneberg, 1943; Bronson et al., 1966). This fact should be kept in mind when trying to count back from the day of birth to the day of conception in order to determine paternity for females in contact with sequential males.

4.3.7 Effects of a foreign male on pregnancy and pup survival

The fertilized embryo is a free-floating entity in the female reproductive tract for the first 4.5-5 days of development. It is during this pre-implantation period that external events can play a role in determining whether a successful implantation will occur. Obvious disturbances to the mental health of the pregnant female — such as erratic lighting, extremes in temperature or humidity, high noise levels, or insufficient food and water — can cause a failure to implant. In addition, there is one other less obvious disturbance that is highly significant in the eyes of the female — the introduction into her cage of a male other than the one with whom she had mated. If the foreign male is not genetically identical to her partner, he can cause a premature termination of the pregnancy through a mechanism which is almost certainly a hormonally induced block to implantation (Bruce, 1959; Bruce, 1968). This pregnancy block is also known as the Bruce Effect (after its discoverer) and it provides an obvious selective advantage by ensuring that females will use their resources only to raise offspring who carry the genes of the intruding male (who is presumably more fit since he has displaced the original mating male). With the previous pregnancy terminated, the female can quickly become pregnant again with her new partner.

It is interesting that females do not recognize males from the same inbred strain as foreign (Bruce, 1968). On the other hand, a pregnancy block is induced in nearly all other cases. These findings indicate that one or more genetic differences are responsible for the distinction between the original and the intruder male, but in addition, they clearly show that the genetic recognition system is highly polymorphic. Further studies with congenic and coisogenic strains have demonstrated conclusively that a major component of this recognition system is the highly polymorphic class I family of genes in the major histocompatibility complex (Yamazaki et al., 1986).

The Bruce effect has important implications for the management of a breeding mouse colony. Quite simply, if a mating event has occurred and one wants to recover live-born offspring from this mating, for the first 5 days that follow, the pregnant female should not be placed either into a cage with a foreign male or in contact with bedding that has been soiled by a foreign male. After this initial stage, there is no longer any problem. In fact, if one wishes to quickly set up a new mating pair, one should be sure to do it before the litter is born. If a foreign male is in the cage at the time of birth, he will normally accept the newborn pups. (Presumably, he "thinks" these pups are his own.) On the other hand, if a male is placed into a new cage that already has newborn pups, he is likely to kill them ("knowing" that he couldn't possibly have been the father).

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