6.1 Background and development of the male and female reproductive tract
The reproductive organs in both sexes are derived principally from embryonic mesoderm and the incorporation of primordial germ cells. There is a small contribution of ectoderm to the formation of the external genitalia. Primordial germ cells, which are the indifferent precursors of sperm and ova, migrate from the yolk sac to the genital ridges early in embryonic development. Occasional abnormal migration leads to abnormal deposition of these cells in extragondal tissues, which can lead to extragonadal teratoma formation in adult animals. Differentiation of the gonads towards the appropriate genders specific organ is a complex process but is generally believed to be initiated by the presence of sex determining factors such as Sry from the Y chromosome.
The tubular structures of the reproductive tract develop from the mesonephric or Wolffian duct and the paramesonephric or Mullerian duct. In the presence of developing testes the production of testosterone and Mullerian inhibiting substance leads to the development of the Wolffian duct system into the seminal vesicles, epididymides and vas deferens from the urogenital sinus and the regression of the Mullerian duct. In the absence of testosterone the Wollfian ducts regress and the Mullerian duct system develops into the uterus, oviduct and vagina (Pritchett and Taft 2007). The caudal genital tract (prostate and ampullary glands) and external genitalia develop in both sexes from the urogenital sinus. At birth the genital tubercle and the anogenital distance are larger in males than females but the external genitalia are still relatively undifferentiated and do not complete development until day 10. The penis and the clitoris derive from an ambivalent genital tubercle with sex differentiation occurring from day 16 of gestation.
Puberty occurs at four to six weeks depending on the mouse strain (Nelson et al. 1990) and environmental cues and is defined by the onset of reproductive competence. In females the formation of the vaginal opening is a sign of the onset of puberty and generally marks the onset of cyclicity, although animals may not be able to sustain a pregnancy immediately. In males puberty is defined by the production of mature sperm although the actual ability to fertilize females may only occur several weeks after mature sperm are first observed (Creasey 2011).
6.2 Female reproductive tract
6.2.1 Sampling technique
Routine analysis of the female reproductive tract should also include analysis of the stage of the oestrous cycle. Although the stage of the oestrous cycle is often assessed on vaginal epithelial morphology, via vaginal smears or histological analysis, it is important to confirm that the rest of the tract including the uterus and ovaries are in synchrony with the appearance of the vagina. For routine analysis immersion in 10% NBF or paraformaldehyde is adequate for fixation of all elements of the tract. In vivo, the stage of the oestrous cycle can be assessed by evaluation of the external genitalia and taking vaginal smears. The vaginal opening varies from a tightly closed slit during dioestrus to being open and surrounded by swollen tissue at proestrus (Champlin et al. 1973; Byers et al. 2012). The stage can be further confirmed by vaginal cytology following the preparation of smears (Byers et al. 2012).
Normal mouse ovaries are very small so to ensure that they are not lost during tissue processing and to aid orientation for sectioning it is best to prepare them in a separate wax block from the rest of the reproductive tract. Biopsy pads or cassette inserts can be used to prevent loss of tissue during processing (Figure 6.1). If the ovary does not need to be weighed or inspected in detail at necropsy then it can be retained in its bursa with the associated oviduct. Longitudinal sections through the middle of the ovary will be sufficient for routine analysis and will also usually yield transverse and oblique sections of oviduct as well as the bursa. For longitudinal sections of oviduct the tip of the uterine horn and oviduct should be dissected and embedded separately (Kittel et al. 2004).
Microscopic analysis of the general populations of corpora lutea and follicles in longitudinal sections of ovary stained with haematoxylin and eosin, in conjunction with evaluation of the rest of the reproductive tract, are generally sufficient to assess whether an animal is cycling normally (Regan et al. 2005). However in some circumstances, for example in the evaluation of the effect of genetic or other manipulations on folliculogenesis, it may be necessary to quantify the number of follicles present within the ovary. In these circumstances a more systematic approach to sampling is required. A variety of stereological approaches based on the evaluation of multiple sections per ovary with correction factors are described and debated in the literature (Tilly 2003). In postnatal studies, ovaries should be harvested from animals at a defined stage in the oestrous cycle (Myers et al. 2004) and after routine processing serial sections made and the number of follicles of different types evaluated in a pre-determined subset of the slides.
The tubular reproductive organs can be prepared in a single wax block. For routine analysis a longitudinal section through the vagina, cervix and body of the uterus with transverse sections from each uterine horn (Kittel et al. 2004) is usually sufficient.
Rarely, it may be necessary to examine specifically the vulva and/or the clitoris and clitoral glands, which lie just cranial to the vulva in female mice. The area of the vulva including the glands can be dissected out and separated from the vagina and embedded to enable longitudinal sections through the area (Ruehl-Fehlert et al. 2003).
Examination of the placenta is not done routinely in phenotypic screening but may be required for specific investigations. Careful selection of appropriate samples is required to allow analysis of the pregnant uterus, placenta and foetal contents (see Blackburn 2000). Sections of uterus should be selected by cutting either side of the gestational sites and embedding the tissue so that longitudinal sections are taken. Serial sections can be made through the site or a single section at the maximum diameter taken for comparison between gestational sites or animals.
6.2.2 Anatomy and histology
The general anatomical structure of the mouse female reproductive tract is similar to most mammals that have multiple offspring and consists of paired ovaries, a duplex uterus with a relatively short body and long uterine horns, cervix and vagina. Significant variations in histological appearance occur with age and stage of the oestrous cycle and an awareness of these changes is important when analysing the tissues. In mature (greater than 5–6 months) fertile mice the oestrous cycle is generally around 4 days (Nelson et al. 1982) if the animal is not mated or pseudopregnant. Between puberty and full maturity the cycle length tends to be longer >5 days (Nelson et al. 1982). From about 12 months (Nelson et al. 1982) (although there is considerable variation depending on strain, nutrition and husbandry factors) the mouse starts to become reproductively senescent, with increases in cycle length and eventual loss of normal cyclicity.
The ovary is contained within a thin-walled bursa (Figure 6.2), which originates from mesovarium that, in turn, attaches the ovary to the peritoneum. The ovarian bursa is usually found embedded in fat just caudal to the kidneys. The bursa is lined on both sides by flattened mesothelial cells and has a thin connective tissue core which contains scattered smooth muscle fibres. Cystic distension of the bursa is common in older mice and needs to be distinguished from cystadenoma. Haemorrhagic ovarian cysts are also common in ageing mice and can become very large (>2 cm in diameter) (Chapter 1, Figure 1.37). Rupture can be associated with significant blood loss, anaemia and even sudden death.
The ovary has a surface epithelial layer composed of flattened to cuboidal epithelial cells (Figure 6.3) derived from the peritoneal lining cells which are attached to a thin basement membrane. The mouse ovary has an outer cortex that contains the follicular structures and corpora lutea and a central medulla which contains stroma and blood vessels (Figure 6.2). The division between cortex and medulla is ill defined and, because of the small size of the ovary, the medulla may not be present in every section. The ovarian stroma contains spindle shaped cells interspersed with collagen and supports the follicles and corpora lutea. The collagen is more dense in the area below the surface epithelium and the region is sometimes called the tunica albuginea although it is poorly defined and may be hard to distinguish in mice.
Figure 6.3 Follicular types in the ovary. (a) Primordial follicles, (b) secondary follicle, (c) antral follicle.
Ovarian follicles are categorized based on the morphological appearance of the associated granulosa cell layers into primordial, primary, secondary, antral and atretic follicles (Figures 6.3 and 6.4) (Myers et al. 2004). Primordial follicles tend to be found towards the subcapsular region of the ovarian cortex and consist of an oocyte surrounded by a single layer of flattened granulosa cells. Polyovular follicles may be seen in young mice but are rare in mature animals (Kent 1960). The growing oocytes are surrounded by a hyalinized layer of glycoproteins secreted by the oocyte (El-Mestrah et al. 2002) called the zona pellucida. In primary follicles the oocyte and zona pellucida is surrounded by a single layer of cuboidal granulosa cells and an outer layer of flattened cells. Secondary follicles have multiple layers of cuboidal granulosa cells in close association with the oocyte, with no antral space. Antral follicles have multiple layers of granulosa cells and a clearly visible fluid-filled antral space (or spaces). The numbers of large antral follicles increase during proestrus and decrease as a result of ovulation during oestrus. In large preovulatory antral follicles, the oocyte is separated from a single antral space by a surrounding layer of granulosa cells (the cumulus granulosa), which will be retained with the oocyte when it is released at ovulation (Figure 6.5). Most follicles do not reach the point of ovulation and either undergo attrition (primordial follicles) or atresia. Oocytes become atretic as a result of apoptosis and so in early follicular atresia fragmentation of the degenerate oocyte and granulosa cells may be seen (Figure 6.6). Remnants of atretic foillicles can be seen as shrivelled eosinophilic remnants of the zona pellucida which stain PAS positive (Figure 6.4) surrounded by theca interna cells. The remaining theca interna cells are sometimes referred to as ‘interstitial glands’.
Figure 6.4 Atretic follicles (thick arrows) and zona pellucida (thin arrow) demonstrated with PAS stain.
The appearance of corpora lutea varies with the stage of the oestrous cycle (Figure 6.7) and whether they are from the current or previous cycles (Figure 6.8). When first forming following ovulation the theca cells (Young and McNeilly 2010), which make up the corpora lutea, have basophilic cytoplasm and are a plump spindle shape, mitoses may be present. The centre of a newly formed corpus luteum may have an irregular fluid filled or haemorrhagic cavity. During metoestrus and dioestrus the cells of the corpus luteum become plumper and may start to accumulate fine lipid vacuoles (Greenwald and Rothchild 1968) reaching maximum size and vacuolation at dioestrus. Fibrous tissue may also be apparent in the centre of the corpus luteum at dioestrus filling what was the haemorrhagic cavity (Westwood 2008). During proestrus the corpora lutea start to degenerate (with vacuolation of cytoplasm), nuclear fragments and neutrophils may be present. Corpora lutea from up to three previous cycles (Greenwald) may be present and are identifiable by being more eosinophilic than the corpora lutea of the current cycle and becoming paler and smaller with time.
Figure 6.7 Corpora lutea at different stages of the oestrous cycle. In (a) prooestrus degenerate cells are present, (b) oestrus there may be central haemorrhage and mitsoses, during (c) metoestrus and (d) dioestrus cells increase in size and become paler and vacuolated.
Figure 6.8 Corpora lutea from current cycle are more basophilic (black star) than those from previous cycles (white star).
The rete ovarii are tubular remnants of the mesonephric ducts, which can be found within the mesovarial fat or within the ovarian stroma at the hilus. The ducts may be single or multiple and lined by cuboidal to columnar epithelium, which may be ciliated. In ageing mice it is common for the ducts to become dilated or cystic (Long 2002).
6.2.3 Oviduct
The oviduct consists of four anatomical regions; intramuscular (uterotubular), isthmus, ampulla and infundibulum (Stewart and Behringer 2012) lined by a folded mucosal surface (Figure 6.9). The light microscopic appearance of the oviductal mucosa does not vary significantly with the stage of the oestrous cycle. The intramuscular portion passes through the tip of the uterine horn and is lined by low columnar cells surrounded by a connective tissue lamina propria with scattered elastic fibres and smooth muscle cells. The remaining oviduct is lined by a single epithelial layer of pseudostratified cells with centrally located nuclei. The cells are a mixture of ciliated and secretory cells with ciliated cells being more common in the infundibulum and ampulla and secretory cells in the isthmus (Yamanouchi et al. 2010; Stewart and Behringer 2012). The ampulla and isthmus have an outer wall of smooth muscle. The isthmus has a thick muscular wall and connects the intramuscular section to the ampulla and is highly coiled in the mouse, so it is common to see multiple cross-sections in standard preparations. The ampulla has an increased lumen diameter compared to the isthmus and connects to the infundibulum which ends in the fimbria in the ovarian bursa.
6.2.4 Uterus and cervix
The uterus consists of a body and two elongated horns (Chapter 1, Figure 1.36). The cranial portion of the uterine body (or corpus) is divided by a midline septum; caudally the body becomes one chamber, which is continuous with the cervix and the vagina. The uterus is composed of an outer loose connective tissue serosal layer (perimetrium), two muscular layers (myometrium) and an inner mucosal layer (endometrium). The myometrium consists of an inner circular and an outer longitudinal layer of smooth muscle fibres separated by a thin vascular connective tissue layer. In the horns and divided portion of the uterine body, the endometrium and endometrial glands are lined by a single layer of columnar epithelial cells, the appearance of which varies with the stage of the cycle (Figure 6.10). The epithelial lining of the undivided portion of the uterine body and cervix is stratified squamous and continuous with the vaginal lining. At the start of oestrus the lumen of the uterus may be mildly distended with fluid, which gradually reduces with time (this normal appearance needs to be differentiated from fluid enlargement due to distal obstruction of the reproductive tract leading to gross distension due to mucometra or hydrometra (Chapter 1, Figure 1.38)). The glandular epithelium starts to degenerate which can be seen by vacuolation and apoptosis of the epithelial cells and there is an absence of mitoses; some infiltrating inflammatory cells may be present. In metoetrus the lumen becomes reduced and the lining epithelium continues to show signs of degeneration, although some mitoses may be present. In dioestrus the uterus is at its thinnest with a narrow slit-like lumen surrounded by a thin endometrium with condensed stromal tissue and low columnar lining cells with no evidence of degeneration although occasional mitoses may be present. In proestrous the uterine lumen becomes dilated by fluid and the lining epithelial cells become tall, columnar with frequent mitoses and the stroma may have an oedematous appearance.
Figure 6.10 Uterine endometrium changes morphology with stage of oestrous cycle. (a) During proestrus the epithelium may contain mitoses and the stroma may be oedematous, (b) epithelium starts to vacuolated and degenerate in oestrus, (c) mitoses are seen towards the end of metoestrus, (d) the epithelium is at its lowest in dioestrus.
A number of changes are commonly seen in the uterus of ageing mice; these include adenomyosis, endometrial hyperplasia and haemangiectasia (Creasey 2011). Adenomyosis is the presence of normal well differentiated endometrial glands and associated stroma within the myometrium, occasionally extending to the serosa, and should not be confused with neoplasia. Endometrial hyperplasia is extremely common in ageing mice, sometimes in association with ovarian cysts. At necropsy, endometrial hyperplasia is recognized as an enlarged and convoluted or segmented appearing uterus (Figure 6.11). The histological appearance can be very bizarre and easily confused with a neoplastic process, although uterine tumours are relatively uncommon in mice. In early or minimal lesions there is an increase in size and number of endometrial glands, which may appear widely separated by oedematous stroma, which in time become more densely collagenous and accompanied by a population of neutrophils. In more advanced lesions the glands become tortuous and cystic . The epithelium may be columnar, with more basophilic cytoplasm and nuclear crowding or may be flattened in cystic areas. Dilatation of blood vessels (haemangiectasia) with associated thrombi may be seen alone or in conjunction with endometrial hyperplasia and can result in sufficient chronic blood loss to cause anaemia.
Figure 6.11 Ageing female mice commonly have varying degrees of endometrial hyperplasia. The uterus can become macroscopically enlarged and convoluted. Microscopically the appearance of hyperplasia, cysts often combined with haemangiectasis and thrombi can be mistaken for neoplasia.
6.2.5 Vagina
The vagina is a tubular structure, which is continuous with the cervical canal and ends caudally at the external genital opening (vulva). The vagina has an outer layer of loose connective tissue (adventitia), two layers of smooth muscle and an inner mucosal layer. The muscular layers are arranged as an inner circular and an outer longitudinal layer. The vaginal (and cervical) mucosal lining consists of a stratified squamous epithelium which changes radically during the oestrous cycle. The cyclic changes can be monitored in vivo by the examination of vaginal smears (Caligoni 2009).
Oestrus is the easiest stage of the cycle to recognize in histological sections (Figure 6.12). During oestrus, the vaginal mucosal is at its thickest (10-12 cells thick) and is covered by a layer of keratinized (cornified) cells. These keratinized cells are flattened, highly eosinophilic and usually anuclear (they may be nucleated early in oestrus). These keratinized cells are shed into the lumen and can be detected on vaginal smears (Figure 6.13). There are no infiltrating neutrophils in the lamina propria or epithelium.
Figure 6.12 Vaginal epithelium changes morphology with stage of oestrous cycle. (a) Proestrus, (b) oestrus, (c) metoestrus, (d) dioestrus.
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