**1. Introduction**

The female reproductive system and processes are dynamic in both humans and rodents, undergoing morphological and cytological changes in response to hormonal signals throughout the estrus (for rodent) or menstrual (for human) cycle. These changes are also observed during pregnancy as well as during menopause or ovarian aging. Mice and rats are animals belonging to the Rodentia order and the Muridae family, and so are referred to as murine. Mice and rat models have been established in preclinical research involving investigations and assessments of reproductive physiological and pathological processes in humans. Their little size as well as the simplicity of their housing and maintenance allow them to be commonly employed in research. Mice and rats are employed as good models of mammalian pathology and physiology given that the use of people and food animals in studies is hampered by economic and ethical reasons.

In experiments relating to human development, the mice models have been mostly utilized to examine the pathogenesis as well as the general attributes in developmental processes. The ample justification for their relevance to human reproductive tract development and functions lies in their hormone action. Just like in humans, their reproductive cycle oscillates periodically through fluctuations in ovarian steroid concentrations (estrogens and progesterone). There also exist these steroid hormone receptors (ER, PR) systems in some of their reproductive organs like the uterus and mammary glands which act as controls to check hormone levels and translate the info into suitable developmental effects. They possess similar reproductive cycle phases as humans. Their reproductive cycle is divided into two developmental phases: the preparatory phase, which is evidenced by high estrogen and progesterone concentrations and occurs throughout proestrus stage. During estrus, this phase leaves the ovarian target tissues prepared for gestation. The second phase occurs in diestrus when progesterone levels drop, and it is characterized by the apoptotic elimination of previous arrangements.

In order to uncover human developmental pathways and gain insight into female reproductive behaviors, developmental scientists have used a number of animals to explore the morphogenetic and molecular mechanisms involved in vertebrate development [1]. Organogenesis and differentiation have been studied extensively in embryonic, fetal, and neonatal mice and rats, with the unspoken assumption that morphogenetic and molecular processes in mice are equivalent to those in humans. This argument is supported by the fact that mice and humans share many developmental features. With a few exceptions, both species share the same range of organs and developmental phases that appear to be equivalent, if not identical [2, 3].

Whereas the data above illustrate that mice research is important to human reproductive biology, the mouse and human reproductive systems have distinct morphogenetic, physiologic, and anatomical distinctions. The lack of information on human organogenesis, particularly in terms of molecular pathways, makes human/ mouse comparisons challenging.

### **1.1 Comparison of the basic anatomy of the reproductive organs of the female mice to humans**

The female mice reproductive systems are made up of two ovaries, two small tightly coiled uterine tubes, two uterine horns, the cervix and the vaginal canal (**Figure 1**). The vagina is a tiny gray tube that connects the bladder to the uterus. In mice, the Mullerian ducts start at the opening of the uterine tube and develop into the female genital tract while in the rats, the ovarian bursa is formed when the ostium forms a complete capsule around the ovary. The uterine horns divide the vagina and reach toward the kidneys. Both the mice and rats possess bicornuate uterus which may accommodate several embryos known as litters. On the other hand, women have simple uterus which have only one compartment for the development of one or two embryo(s) (**Figure 1**). Ovaries in humans are little lumpy glands that sit at the terminals of the uterine horns and are connected to the uterine horns by oviducts.

*Our Clear-Cut Improvement to the Impact of Mouse and Rat Models in the Research Involving... DOI: http://dx.doi.org/10.5772/intechopen.106858*

**Figure 1.** *Anatomical feature of the reproductive system of a female rat and human.*

#### *1.1.1 Mice uterus and cervix in comparison with humans*

The human uterus is simplex and appears in form of a large pear-like shape with a unique cavity which measures about 8 cm when assessing it from one cornu to the other cornu and 5 cm in width (**Figure 1**). Nevertheless, steroid hormone levels as well as pregnancy status extensively determines the thickness of the uterus. Other factors that affect uterus thickness include the existence of leiomyomata. On the other hand, mouse uterus is bicornuate having two cornua that unite distally to create a singular body (corpus), but in rats, the uterus is duplex, with twin uterine horns that are partially linked at the caudal end. A part of the broad ligament known as mesometria suspend the mouse uterus horns from the dorsal wall and hold them in place. These ligaments encircle the blood and lymphatic vessels as well as many nerves. Similarly, from the lateral pelvic side walls, the human uterus is also suspended by comparable ligaments. The body of the uterus both in humans and mice is made up of the caudalcervical and cranial-fundal sections. The caudal-cervical and cranial-fundal regions of the uterus are also found in mice. However, the cranial portion of the body of mouse uterus is separated by a median septum into two chambers. On the other hand, rat uterus is partly connected and has no complete body. Nonetheless, it has a unique cervix with two uterine horn canals that are distinct. Both dorsally and ventrally, mice and rats have a continuous cervix and vaginal walls but not laterally. The lateral vaginal walls continue on both sides forming deep fornices. But then, in humans, the cervix (ectocervical portion) protrudes into the vaginal canal, having wide posterior and anterior fornices and thinner lateral fornices.

#### *1.1.2 Comparison of the oviduct (uterine tube)*

The oviduct, generally termed the uterine tube in mice and rats is a complex tinycurled tube that links to the periovarian area. The mesotubarium helps to suspend the oviduct to the dorsal body wall, and is connected to the mesovarium, uterine mesometrium and ovarian bursa. Whereas the human oviduct is a muscle-lined straight tube that is about 12 cm long. The human oviduct, like that of rodents, is connected to the respective ovary by a broad ligament that runs from the uterine cornu (horn) (**Table 1**). In both the mice, rat and human oviducts are found the intramuscular segment, the isthmus, the ampulla, the infundibulum, and a fimbriated terminal [4].


#### **Table 1.**

*Comparison of the female reproductive system of rat and human.*

Inside the uterine wall is found the oviduct's intramuscular section which continues into the dorsolateral side of uterus wall in rodents, forming the colliculus tubarius, a modest extension into the uterine cavity. The dimension of this extension differs although it is usually a smaller number (1mm in length). Depending on uterine size, the human oviduct's intramuscular section length varies and reaches laterally from the superior corner of the cornu until it arises from the uterus wall. A brief segment of the oviduct, the isthmus runs laterally from the uterus wall to the ampulla (**Figure 1**). This segment is significantly more firmly twisted in mice than in humans [5]. The ampulla is a dilated section that connects the oviduct's infundibulum to the isthmus. From the infundibulum, the oviduct of mice grows a fimbriated side that emerges into periovarian area and the peritoneal cavity close to the ovary. In humans, one of these fimbriae joins the ovary to the oviduct.

#### **1.2 Sexual maturation in female murine (rat and mice) compared to humans**

In adult female mice, reproduction is comprised of a sequence of neurological and hormonal changes that interact to allow the creation of offspring. The connection between the anterior pituitary and ovarian hormones, as well as placental hormones, is involved in this process (during pregnancy). The hypothalamus, in particular, plays a key role in controlling the anterior pituitary's ability to manufacture and

*Our Clear-Cut Improvement to the Impact of Mouse and Rat Models in the Research Involving... DOI: http://dx.doi.org/10.5772/intechopen.106858*

secrete gonadotropin hormones (Follicle stimulating hormone, FSH and Luteinizing hormone, LH). FSH enhances the development of gametes in both male and female animals, whereas LH stimulates the release of estrogen and progesterone (gonadal hormones) in females as well as androgen in males. Lactation is regulated by prolactin, an anterior pituitary hormone.

Secondary sexual traits and the correct functioning of the genital tract are maintained by gonadal hormones, that also work somewhat on central nervous system to ensure effective coupling [6]. Each part develops at a varying rate, and normal sexual maturity in rats relies on a complex and poorly known interaction among them all.

Sexual maturity normally correlates with increasing circulating gonadotropin titers after 4 weeks of age. In contrast to humans, the specific moment at which maturity occurs is quite varied, therefore such a comment must be interpreted in the context of the measurement used to determine when "sexual maturity" has been attained (**Tables 2** and **3**). Vaginal introitus and a cornified vaginal smear, both estrogen-dependent, are the earliest detectable indications of puberty in mice and rats. Other markers of sexual maturity, such as mate readiness, the capacity to conceive and carry a litter to term, and maybe sexual maturity as measured by the ability to produce weanling-age young, have a more convoluted hormonal foundation. The vaginal opening can happen as early as day 26 and is usually complete by the 7th weeks (**Tables 2** and **3**). The first vaginal cornification occurs between 24 to 120 hours after the vaginal opening has been created, however this is very variable. Furthermore, estrus (the urge to mate) does not always occur on a regular timetable, which can contribute to unpredictability and make it difficult to use as an experimental tool.

Regardless of how it is measured, the process of achieving sexual maturity is a very varied one. According to the timing of vaginal opening, inbred mice strains generally mature by 7 weeks of age, however some researchers have observed a median age of 35 days and a range of 26 to 49 days in C57BL/6 and BALB/c strains [7]. Vaginal opening has been discovered as early as 24 days in certain experimental animals. Two key drivers to such variety are genetic background and season. Many studies have discovered


#### **Table 2.**

*Sexual maturity and ovulation in rat and human.*


**Table 3.**

*Difference between human endometrial cycle and mouse estrous cycle.*

that there is some inter-strain variability. For example, Drake et al. [8] discovered that vaginal introitus occurs at a younger age in the summer than in the winter. He was able to account for a considerable percentage of the linked variation by matching a sine curve to the data and accounting for seasonal fluctuations. The season of the year also has a huge impact. Experimental exposure to cold delays vaginal opening, the first cornified smear, and the first typical estrus.

#### **1.3 Relationship between menstrual cycle and estrous cycle**

The menstrual cycle, as well as the estrus cycle, is controlled by the regular intermittent fluctuations in the mean value of estradiol (E2) and progesterone (P4) gonadal steroid hormones in endocrine fashion (**Figure 2**). In tissues such as the uterus and ovaries, E2 and P4 receptors (ER, PR) track the concentrations of these hormones and convert the information into timed developmental feedbacks.

The key integrator of both the menstrual and estrous cycles is gonadotropin releasing hormone (GnRH), a neurohormone discharged in pulsatile manner by the hypothalamus. The GnRH, when released is transported via the hypophyseal tract to the anterior pituitary gland that causes the production of FSH and LH gonadotropic hormones (**Figure 3**). These hormones are conveyed in the blood vessels to the ovaries to cause development of follicles and resultant production of E2. In this regard, FSH has stronger effects. During the pre-ovulatory stage, E2 confers a positive feedback effect on GnRH, FSH and LH production thereby increasing its own concentration, while it elicits a negative feedback effect during the post-ovulation period [9].

Two contrasting developmental phases exist in both menstrual and estrous cycles. The first is the follicular phase (in humans) also known as pro-estrus phase (in animals). It is a preparatory stage evident by the raised E2 and P4 levels (**Figures 2** and **4**). These steroid hormones initiate the proliferation of the endometrium and growth of

*Our Clear-Cut Improvement to the Impact of Mouse and Rat Models in the Research Involving... DOI: http://dx.doi.org/10.5772/intechopen.106858*

**Figure 2.**

*The 4–6 day mouse reproductive cycle (left panel) is compared to the 28-day human menstrual cycle (right panel). In this diagram, average fluctuations of estradiol, progesterone, LH, and FSH (Maeda et al., 2000).*

#### **Figure 3.**

*Activities of reproductive hormones on the ovaries in estrus phase. The hypothalamus through E2 positive feedback loop secretes GnRH which consequently increases FSH concentration. The FSH reaches the ovaries to cause the development of the follicles which secrets E2. The increased E2 levels cause the proliferation of endometrial lining and inhibition of DA. The inhibition of DA releases PRL from DA suppression. PRL maintains CL formed after ovulation. After ovulation, E2 confer a negative feedback effect on the secretion of GnRH, FSH and LH. CL formed produces high concentrations of P4 and low E2 levels leading to secretory activity in oviduct and endometrium. The low E2 concentration releases the GnRH and DA from inhibition. DA suppresses PRL leading to CL regression and consequent low levels of P4. GnRH = gonadotropin releasing hormone, LH = luteinizing hormone, FSH = follicle stimulating hormone, P4 = progesterone, E2 = estrogen, PRL = prolactin, DA = dopamine and CL = corpus luteum.*

#### **Figure 4.**

*The hormonal levels in each phase of mouse reproductive cycle. Diestrus is the end of luteal phase; pro-estrus is the beginning of follicular phase. DE = diestrus, PE = pro-estrus, E = estrus, ME = metestrus, GnRH = gonadotropin releasing hormone, E2 = estradiol, P4 = progesterone, PRL = prolactin, DA = Dopamine, LH = luteinizing hormone, FSH = Follicle stimulating hormone.*

blood vessels and prime the reproductive tissues for pregnancy [10]. The second is the luteal phase (in humans) also identified as metestrus phase (in animals). It begins as E2 and P4 concentrations decline and manifests as apoptotic breaking up of the former preparations in addition to resorption of the uterine endometrium. The most outstanding peculiarity of the estrous cycle is the steady E2 surge in late diestrus to pro-estrus. This event marks the border between the two developmental phases of the cycle and signifies the beginning of a fresh cycle [11].

#### **1.4 Difference between menstrual cycle and estrous cycle**

Mice and rats go through estrous cycles, of which if fertilization failed to occur during the cycle, resorption of endometrium occurs. Humans on the other hand, experience menstrual cycle of which their endometrium is shed during the course of menstruation if fertilization and conception failed to occur. One more difference is in their sexual behavior during ovulation [12]. Female mice and rats are normally sexually receptive and active solely at some point in their estrous cycle such as during late proestrus and in estrus phase which are the phases where ovulation occurs. This is known and referred to as "heat period." Females of species with menstrual cycle, such as humans, on the other hand, can be sexually receptive and active all over the course of their cycle. Humans also undergo covert ovulation with no visible outside displays of sexual receptivity unlike the mice. Rats, on the contrary frequently display unmistakable outward receptivity to demonstrate estral receptivity at ovulation [13].

*Our Clear-Cut Improvement to the Impact of Mouse and Rat Models in the Research Involving... DOI: http://dx.doi.org/10.5772/intechopen.106858*

A classic menstrual cycle in humans lasts about 28 days, with ovulation taking place at 14th day (**Figure 2**). The estrus cycle is considerably shorter than the menstrual cycle, occurring within 4 to 6 days [14, 15].

Additional key difference between the cycles, aside the overall period necessary for a full cycle, remains that the E2 and P4 peaks are classically disconnected in human menstrual cycle, while they intersect in rodent estrous cycle at the pro-estrus phase [16]. Rodents are furthermore disposed to estrous cycle interference from sensitivity to external or environmental cues such as light, temperature, stress and other factors than the humans are [17].

#### **1.5 The mouse estrous cycle and hormonal changes**

Hormonal changes always and frequently manifest as regular alterations in the morphology and cytology of the animal's reproductive tract.

The estrus cycle in mice is divided into 4 distinctive phases – pro-estrus, estrus, metestrus and diestrus. The pro-estrus phase corresponds to follicular phase in humans. At about midday of the beginning of the pro-estrus phase, there exists a significant surge of estradiol (E2) triggering a fast peaking of the LH and FSH in the evening of pro-estrus and an increased progesterone (P4) secretion [11]. As in humans, the gonadotropin surge prompts ovulation and subsequent formation of corpus luteum. This high concentration of E2 inhibits dopamine (DA) and simultaneously increases the concentration of prolactin (PRL) by relieving it from dopamine inhibition (**Figure 3**). All hormones come back to starting levels when ovulation occurs in estrus.

The PRL and P4 levels increase markedly at the early post-ovulation phase (i.e. late estrus) and drops abruptly in metestrus phase (**Figure 4**). The PRL is responsible in maintaining the corpus luteum. During the estrus and metestrus phases, the corpus luteum secretes P4 and to a lesser extent E2 as well as inhibin [14]. All these hormones have combined negative feedback on the GnRH, FSH and LH [9]. By the late diestrus phase, the corpus luteum regresses due to decrease in PRL levels. This effect consequently leads to a decline in E2, P4 and inhibin levels. All these releases the hypothalamus and anterior pituitary from the negative feedback effects of these hormones [11] and initiates the start of a new phase

#### **1.6 Characterization of menstrual cycle using different phases of estrous cycle**

#### *1.6.1 Pro-estrus phase*

Pro-estrus phase lasts for 24 hours (approximately 1 day) in mice and rats [15]. This phase parallels the pre-ovulatory day of menstrual cycle. For example, E2 concentration rises and confers a positive feedback mechanism on GnRH release. At mid pro-estrus phase, E2 concentration reaches its peak to induce the LH and the FSH surge resulting in ovulation while it confers an inhibitory effect on DA to release PRL suppression [17]. During this phase, P4 and PRL levels begin to rise (**Figure 4**). One or several follicles of the ovaries start(s) to grow and mature (**Figure 3**).

The main characteristics of vaginal cytology of this phase is the existence of large or small, rounded epithelial cells that may be nucleated or enucleated. The cells are fairly of uniform appearance and size. They are usually seen in cohesive sheets, clusters, or strands. The appearance might sometimes not be observed particularly in hypocellularity samples, therefore should not be taken as a yardstick in determining pro-estrus. Sometimes no leucocytes will be seen, however, leucocytes can be found in early pro-estrus.

Moderately low amounts of large epithelial cells plus cornified cells may as well be detected. As the cycle nears estrus, abundant cornified cells will be present (**Table 4**). The presence of high amounts of cornified cells or low numbers of leucocytes should not impede the identification of pro-estrus especially when the usual features of the smear are the small, round epithelial cell population. There might be presence of the secretion in the smear. Visual observation of the vulva will reveal wide opened vagina which will be moist, and the tissues appears deep pink or red. Striations will be seen in both the ventral and dorsal lips of the vulva. The lips may appear swollen.

#### *1.6.2 Estrus phase*

Estrous phase lasts between 12 - 48 hours (approximately 2 day) and signifies the beginning of the luteal period in humans [18, 19]. Estrus is the stage when the female mice remain sexually receptive ("on heat" or "in heat"). Under regulation of gonadotropic hormones, estrogen secretions exert their biggest influence. Throughout the morning of estrus E2 levels remains elevated conferring inhibitory effect on DA. The action of high E2 on DA releases PRL from DA suppression. PRL is important in maintenance of CL after ovulation. During the afternoon, E2 falls back to basal levels and the P4 concentration on the other hand rises and peaks (**Figures 3** and **4**). The P4 concentration is important for the secretory activities in the endometrium. The high level of P4 with corresponding low concentrations of E2 inhibits GnRH and FSH and releases the DA from suppression (**Figure 4**). DA then inhibits the PRL causing CL to regress which in turn decreases the concentration of P4 at late estrus.

This phase is typically recognized by the presence of abundant cornified squamous cells. These cells appear in clusters and are of irregular shapes. They have no visible nucleus, and their cytoplasm looks granular. Abundant bacterial cells may be seen. The bacterial cells may stick to the cells or appear freely in the background of the smear. During early and mid-estrus phase, no leucocytes are observed however, can be seen during late phase. There will be predominance of cornified cells (75%) with epithelial cells (<25%) (**Table 4**). Cells can appear in clusters or scattered. During physical visual inspection, the vagina will appear similar to pro-estrus, but it will be less pink or red, less moist but striations might be very prominent for some animals. The vulva lips will be swollen.

#### *1.6.3 Metestrus phase*

Metestrus phase marks the mid of luteal phase (post-ovulation) in humans. During this phase, the signs of E2 and P4 stimulation subside. The decline in the plasma levels of P4 stimulates the hypothalamus to secrete GnRH and consequently the gonadotropic hormone, FSH. The FSH in the ovaries starts the development of preantral follicle which secretes E2 (**Figures 3** and **4**). The Low levels of P4 also lead to resorption of endometrial lining. This phase typically is brief occurring within 24 hours [18] or may last for 1–3 days [16], personal experience in our lab, data not published. In this stage, there is presence of leucocytes in combination with few

*Our Clear-Cut Improvement to the Impact of Mouse and Rat Models in the Research Involving... DOI: http://dx.doi.org/10.5772/intechopen.106858*


#### **Table 4.**

*Vaginal smear cytology and mice behavior during estrous.*

cornified squamous epithelial cells (**Table 4**). In early metestrus, leucocytes are sometimes interspersed or may be tightly clumped together around the cornified cells; the leukocyte cells may equal the quantity of the cornified cells or may be less. At mid-metestrus, leucocytes become higher in number than the cornified cells [14] and the smear might be extremely dense and cellular. By late metestrus, the number of cornified cells decreases with a correspondingly reduction in smear cellularity displaying conversion to diestrus phase. Visual observation of the vulva is characterized by pale coloration. The vaginal canal will be completely sealed and dry. It may additionally be sloughed with white cellular debris.

#### *1.6.4 Diestrus phase*

This phase marks the boundary between luteal phase and follicular phase and lasts between 55–57 hours (approximately 3 days) (**Table 4**). If pregnancy fails to occur, the diestrus stage ends with the recession of the CL. In the late stage of diestrus, the hypothalamus through E2 positive feedback loop secretes GnRH and consequently FSH (**Figures 3** and **4**). The FSH in the ovaries causes the development of the follicles from preantral follicle to early Graffian follicle which secretes E2. The increased E2 levels cause the endometrial lining to begin to proliferate. The vaginal cells consist predominantly of leucocytes at the early phase and with nucleated cells but no cornified cells at the late phase. Visual observation will reveal vaginal opening which may be actually moist. The orifice will be slightly opened or totally closed in some animal. There will be no tissue swelling or striation.

#### **1.7 Importance of rodent (mice and rats) in research**

Because better molecular tools to modify the mouse genome were available in the past, the mouse was frequently used instead of the rat. Recent improvements in genetic methods for creating knockout rat models promise to break down these obstacles, potentially allowing rats to be used in a wider range of scientific studies. In the end, the rodent model of choice is determined by which species best mimics the symptoms and illness process seen in humans. It's evident that rats aren't just big mice, and that each species has advantages and disadvantages that vary depending

on the process or gene under investigation. It is very important to adopt the right paradigm for translational medicine because a lot of money is spent exploring medications and cures that ultimately fail at various levels of pre-clinical and clinical trials. One explanation for this is that animal trial results do not always correctly represent human outcomes.
