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Home , Corpus luteum, Cumulus oophorus, Follicular antrum, Folliculogenesis, Ovary, Theca externa

PART 3: FEMALE

CONTENTS

1. Normal female reproductive tract histology 2. Endocrine control of the oestrous cycle 3. Morphological changes during the oestrous cycle 4. Normal background variation of structure and common spontaneous lesions 5. Common morphological responses to endocrine disruption 6. References and bibliography

INTRODUCTION

i. The female laboratory rat, like most placental , demonstrates intrinsic reproductive cyclicity, characterised by the regular occurrence of an oestrous cycle. During this cycle, numerous well defined and sequential alterations in reproductive tract histology, physiology and cytology occur, initiated and regulated by the hypothalamic-pituitary-ovarian (HPO) axis. The changes in vaginal cytology are discussed separately in more detail in Part 5 of the Guidance Document. ii. The oestrous cycle consists of four stages, termed prooestrus, oestrus, metoestrus (or dioestrus 1) and dioestrus (or dioestrus 2). Because rats are continuously polyoestrous (i.e., cycle constantly throughout the year), dioestrus is immediately followed by the prooestrus phase of the next cycle. Anoestrus, a period of reproductive quiescence between oestrous cycles, is thus not usually observed in healthy, cycling female rats. Oestrous cyclicity only ceases during , , and , although a fertile postpartum oestrus does occur within 24 hours after . iii. Sexual maturity in female rats usually occurs between 30 and 50 days of age. Kennedy and Mitra (1963) reported the mean age of in female rats, based on the occurrence of vaginal opening (VO) and first oestrus, as 38 days. Recent studies evaluating the rodent pubertal female assay have recorded similar mean VO ages in control rats of 32 and 35 days (Hyung et al, 2002; Goldman et al, 2000). Following puberty, the oestrous cycle occurs regularly every 4-6 days for a variable proportion (25-70%) of the animal’s lifespan, depending on the strain of rat. Sprague-Dawley rats, for example, demonstrate oestrous cycle abnormalities as early as 6 months of age. In contrast, Fischer 344 rats show far greater reproductive robustness, typically cycling normally until 15 to 18 months of age. ix. The length of each stage of the oestrous cycle, as reported by Mandl (1951), and Long and Evans (1922), is shown in Figure 1.1. These workers found that within a given population of normally cycling rats, dioestrus showed the greatest variation in duration and thus exerted the most influence over oestrous cycle length.

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Mandl (1951)

Long and Evan (1922)

Figure 1.1 – Duration (in hours) of the four stages of the oestrous cycle. Note the short duration of metoestrus.

1. NORMAL FEMALE REPRODUCTIVE TRACT HISTOLOGY

Ovary

1.1 The paired of the rat are grape-like structures that vary in gross appearance and size, depending on the stage of the oestrous cycle. The subgross of the rodent is shown in Figure 1.2. Covering its surface is a single layer of modified peritoneal , the ovarian surface (OSE), which is continuous with the broad () that supports the ovary. The OSE of a single ovary can range from squamous to cuboidal, columnar or pseudostratified columnar in type; this regional variation in OSE morphology accompanies the cyclical changes that occur within the underlying ovarian parenchyma during the oestrous cycle (Figures 1.3a and 1.3b).

Figure 1.2 – Subgross anatomy of the normal rodent ovary (mouse, H&E x4). The cortex (C) contains numerous follicles at various stages of maturation. The medulla (M), which is not always present in histological sections, contains lymphatics, and numerous blood vessels.

C cortex

M medulla

CL

F developing follicles

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Figure 1.3a – Ovarian surface epithelium (OSE) – columnar type (rat, H&E x40).

OSE ovarian surface epithelium TA tunica albuginea C cortex

Figure 1.3b – Ovarian surface epithelium (OSE) – simple squamous type (rat, H&E x40).

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1.2 The mesovarium completely encloses each ovary and within a single compartment, the ovarian bursa (Figure 1.3c). In rodents, the ovarian bursa communicates with the via a small slit-like opening.

Figure 1.3c – (RO) and ovarian bursa (OB) (rat, H&E x10).

1.3 The ovarian stroma forms the body of the ovary and is composed of spindle-shaped, fibroblast-like cells and delicate collagen fibres admixed with ground substance. The stroma directly beneath the OSE is dense and fibrous, and forms a narrow and variably distinct zone termed the tunica albuginea. The ovarian stroma beneath the tunica albuginea is divided into a peripheral cortex and central medulla (Figure 1.2), although the latter is not always visible in histological sections of ovary.

1.4 The rete ovarii may be observed histologically within the rodent ovary. This structure arises from cells of mesonephric origin which migrate into the developing during embryogenesis. In the adult rat, the rete ovarii is composed of several groups of anastomosing tubules embedded within the ovarian stroma and lined by a cuboidal or columnar epithelium (Figure 1.3c).

1.5 In sexually mature rats, the cortex contains numerous follicles at various stages of development. Five stages of follicular maturation () are described:

i. Primordial follicle – This represents the earliest stage of follicular development. Primordial follicles form during early foetal development and are typically located within the peripheral cortex, just beneath the tunica albuginea. Each primordial follicle consists of a primary surrounded by a simple squamous follicular epithelium. Envelopment of the primary oocyte by follicular cells arrests development of the at the first meiotic division. During each oestrous cycle a cohort of “resting” primordial follicles starts to develop into primary follicles; this process occurs occurs independently of hormonal stimulation up until the formation of early tertiary follicles. ii. Primary follicle – The squamous follicular cells surrounding the primordial follicle differentiate into a single layer of columnar cells, forming a primary follicle (Figure 1.4).

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Figure 1.4 – Primary (A) and early secondary follicle (B) (rat, H&E x40).

O primary oocyte

ZG developing zona granulosa

TA tunica albuginea

OSE ovarian surface epithelium (cuboidal)

iii. Secondary follicle – Proliferation of the columnar cell monolayer results in the formation of a multilayered zone of granulosa cells, the zona granulosa, around the oocyte. This is accompanied by the development of a thick and acid proteoglycan coat, the , between the oocyte and the zona granulosa (Figures 1.4 and 1.5). As the secondary follicle continues to grow, multiple fluid-filled spaces form within the zona granulosa; this stage is termed a vesicular follicle. Ovarian stromal cells surrounding the developing follicle become arranged into concentric layers and form the folliculi, or theca (Figures 1.5 and 1.6). This layer is separated from the zona granulosa by a basement membrane.

iv. Tertiary follicle – The cystic spaces within the zona granulosa coalesce and form a large central cavity, the . This cavity is filled with fluid, the liquor folliculi, and surrounded by the zona granulosa. The primary oocyte is eccentrically positioned within the tertiary follicle and resides within a mound of granulosa cells, called the , that protrudes into the antrum. The granulosa cells immediately surrounding the oocyte are termed the corona radiata (Figure 1.7).

The theca of the tertiary follicle is divisible into two zones: a and . The theca interna consists of polygonal cells with vacuolated cytoplasm and open faced, vesicular nuclei. These cells demonstrate the typical ultrastructural characteristics of producing cells (e.g. numerous cytoplasmic lipid droplets, large numbers of mitochondria, and an extensive smooth endoplasmic reticulum), and are the main site of synthesis of (a steroid intermediate). In contrast, the cells of the theca externa are spindle-shaped and merge with the surrounding ovarian stroma; they serve no endocrine function.

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Figure 1.5 – Secondary follicle (mouse, H&E x20).

O primary oocyte ZG zona granulosa

ZP zona pellucida TF theca folliculi

VS vesicular spaces

Figure 1.6 – Vesicular follicle (mouse, H&E x20). Vesicular spaces (VS) are clearly visible within the zona granulosa (ZG).

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Figure 1.7 – Tertiary follicle (rat, H&E x20). The large follicular antrum (FA) and eccentrically positioned primary oocyte (O) characterise this stage.

O primary oocyte CO cumulus oophorus CR corona radiata FA follicular antrum ZG zona granulosa TF theca folliculi

v. Preovulatory (Graafian) follicle – A small number of tertiary follicles enter a preovulatory stage and undergo further morphological changes. The follicular antrum continues to enlarge, causing attenuation of the surrounding zona granulosa. Degeneration of the granulosa cells of the cumulus oophorus occurs; this causes the primary oocyte to detach from the zona granulosa and float freely within the follicular antrum (Figure 1.8). The primary oocyte completes the first meiotic division just prior to and forms the secondary oocyte.

Figure 1.8 – Preovulatory (Graafian) follicle (rat, H&E x40). The primary oocyte (O) floats freely within the follicular antrum (FA).

O primary oocyte ZP zona pellucida CR corona radiata FA follicular antrum ZG zona granulosa

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1.6 Following extrusion of the secondary oocyte from the Graafian follicle, the granulosa and thecal cells of the follicle remnant undergo hypertrophy and, to a lesser extent, hyperplasia (Figure 1.9). This process, termed lutenisation, occurs under the influence of lutenising (LH) and , the two major luteotrophic in rodents. Lutenisation is accompanied by degeneration of the basement membrane separating the theca interna and zona granulosa, and infiltration of the postovulatory follicle by blood vessels from the theca interna. The resulting mature corpus luteum (“yellow body”) is a large eosinophilic structure that may bulge out from the ovarian surface or obscure the ovarian corticomedullary junction, depending on its location (Figure 1.10).

Figure 1.9 – Corpus luteum (rat, H&E x40). The luteal cells (LC) comprising the corpus luteum are plump and polygonal; they contain large nuclei and moderate amounts of eosinophilic cytoplasm. Cytoplasmic vacuoles form within luteal cells as the corpus luteum matures and subsequently degenerates. Numerous blood vessels (BV) are present, consistent with its function as a temporary .

Figure 1.10 – Corpora lutea (CL) (rat, H&E x10). Note the marked protrusion of these large postovulatory follicles beyond the surface of the ovary. Another pair of corpora lutea are present within the body of the ovary.

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1.7 Each corpus luteum matures during the oestrous cycle in which it is formed before regressing over the course of several subsequent cycles. Consequently, at least three sets of corpora lutea are present within the ovaries of normally cycling rats. Degenerating corpora lutea progressively shrink in size and are characterised by increased amounts of fibrous tissue and yellow-brown lipofuscin pigment (Figure 1.11). The fibrous tissue mass that constitutes the corpus luteum during the final stages of regression is termed the (“white body”); this undergoes complete regression in the rat, leaving no fibrous tissue remnant within the ovary.

Figure 1.11 – Degenerating corpus luteum (rat, H&E x20). Note the invading fibroblasts (F) and lipofuscin pigment (L).

1.8 Only a small number of primordial follicles within the cohort progress through folliculogenesis to form Graafian follicles and ovulate. The remainder undergo follicular degeneration, or atresia, at various stages during follicular maturation. In rodents, degenerating tertiary follicles (Figures 1.12 and 1.13) eventually give rise to interstitial glands within the ovarian stroma, although these are more obvious in the mouse than the rat. These glands are composed of aggregates of theca interna cells, often arranged around remnants of degenerate zonae pellucida (Figure 1.14). They are transitory structures, eventually breaking up into small clusters of interstitial cells that become scattered throughout the medulla.

Figure 1.12 – Degenerate (atretic) tertiary follicle (TF) (rat, H&E x10). A viable vesicular follicle (VF) is also present.

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Figure 1.13 – Degenerate (atretic) tertiary follicle (rat, H&E x40). Pyknotic nuclei and karyorrhectic nuclear debris are scattered throughout the degenerate zonular granulosa (ZG).

TF theca folliculi FA follicular antrum ZG degenerating zona granulosa

Figure 1.14 – Interstitial glands (mouse, H&E x20). Theca interna cells surround remnants of degenerate zonae pellucida (ZP).

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Uterus

1.9 The rat is duplex, i.e. it comprises two that join together and open into the via two separate cervices. The basic histological organisation of the uterus is shown below in Figures 1.15:

Figure 1.15 – Uterine horn (rat, H&E x10, longitudinal section).

En Ep surface epithelium LP lamina propria G endometrial gland

My C circular layer (inner) L longitudinal layer (outer)

Pe

Figure 1.16 – Endometrium (rat, H&E x20). The variation in surface epithelium height is an artefact of sectioning.

Ep surface epithelium BV LP lamina propria P pigment G endometrial gland

The inner mucosa, or endometrium, consists of a surface columnar epithelium overlying a thick lamina propria containing numerous blood vessels and endometrial glands. The middle muscular layer, or myometrium, is composed of an inner circular and outer longitudinal layer. The myometrium is covered by the perimetrium, a thin connective tissue layer overlain by a simple serosa. Variable numbers of lymphocytes and other leucocytes are present within the superficial lamina propria of the endometrium. Pigmented stromal cells, containing fine to coarse green-brown granules composed of ferritin, haemosiderin and lipofuscin, may also be observed within the lamina propria of postpartum and mature rodents (Figure 1.16).

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Vagina

1.10 The basic trilaminar arrangement of tissues observed in the uterus is conserved in the vagina, which consists of an inner mucosa, middle muscularis and outer adventitia (Figures 1.17 and 1.18). The vaginal mucosa comprises a lamina propria covered by a stratified squamous epithelium (Figure 1.19). The mucosa undergoes profound cyclic changes over the course of the oestrous cycle; these alterations are discussed in more detail below. The smooth muscle bundles that form the muscularis are also disposed in inner circular and outer longitudinal layers, although these are ill-defined in comparison with those forming the myometrium. The outer longitudinal layer of the muscularis merges with the adventitia, a thin outer connective tissue layer.

Figure 1.17 – Transverse section through vagina and (rat, H&E x4).

V vagina U urethra Lu vaginal lumen Ep surface epithelium

Figure 1.18 – Transverse section through vagina (rat, H&E x10).

Lu vaginal lumen Ep surface epithelium LP lamina propria M muscularis Ad adventitia

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Figure 1.19 – Transverse section through vagina (rat, H&E x20). During prooestrus, the consists of four layers (Table 3.1). The stratum granulosum (SG) comprises the superficial 2-3 cell layers immediately beneath the stratum corneum (SC). The stratum germinativum (SGerm) is the only layer that is present throughout the oestrous cycle.

SM stratum mucification

SC stratum corneum

SG stratum granulosum SGerm stratum germinativum

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2. ENDOCRINE CONTROL OF THE OESTROUS CYCLE

Introduction

2.1 The cyclic changes that occur in the female reproductive tract are initiated and regulated by the hypothalamic-pituitary-ovarian (HPO) axis. Although folliculogenesis occurs independently of hormonal stimulation up until the formation of early tertiary follicles, the gonadotrophins lutenising hormone (LH) and follicle stimulating hormone (FSH) are essential for the completion of follicular maturation and development of mature preovulatory (Graafian) follicles.

The typical reproductive hormonal patterns observed over the course of the rat oestrous cycle are shown schematically in Figure 2.1 below. The sites of production and key functions of these hormones are summarised in Table 2.1.

O oestrogen PL prolactin LH lutenising hormone FSH follicle stimulating hormone P

Figure 2.1 – Reproductive hormone fluctuations during the rat oestrous cycle. Oestrogen and lutenising hormone levels peak during prooestrus. Ovulation follows approximately 10-12 hours later and coincides with the progesterone peak. Follicle stimulating hormone peaks twice in the rat, with the secondary FSH peak occurring at the time of ovulation or shortly after (see text for further details).

D dioestrus PO prooestrus

Oe oestrus M metoestrus

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Pituitary gonadotrophin secretion drives follicular maturation and oestrogen secretion

2.2 Levels of LH and FSH begin to increase just after dioestrus. Both hormones are secreted by the same secretory cells (gonadotrophs) in the pars distalis of the (adenohypophysis). FSH stimulates development of the zona granulosa and triggers expression of LH receptors by granulosa cells. LH initiates the synthesis and secretion of androstenedione and, to a lesser extent, by the theca interna; these are utilised by granulosa cells as substrates in the synthesis of oestrogen. Pituitary release of gonadotrophins thus drives follicular maturation and secretion of oestrogen during prooestrus.

2.3 Gonadotrophin secretion by the anterior pituitary is regulated by lutenising hormone-releasing hormone (LHRH), produced by the . LHRH is transported along the axons of hypothalamic neurones to the , where it is secreted into the hypothalamic-hypophyseal portal system and transported to the anterior pituitary. The hypothalamus secretes LHRH in rhythmic pulses; this pulsatility is essential for the normal activation of gonadotrophs and subsequent release of LH and FSH.

Ovulation is triggered by an oestrogen-mediated preovulatory LH surge

2.4 The increase in oestrogen observed during prooestrus initiates several characteristic morphological changes in the uterus and vagina (Table 2.1). This rise in oestrogen also suppresses release of LHRH by the hypothalamus, as well as directly inhibiting pituitary secretion of both LH and FSH. Negative feedback control of pituitary FSH secretion is also achieved by the inhibin, produced by the granulosa cells of the maturing follicle. The various hormonal interactions of the HPO axis are summarised in Figure 2.2 below.

2.5 Oestrogen levels rise during the morning, peak around midday and then fall during the afternoon of prooestrus. Once peak levels are reached, the inhibition of LHRH and gonadotrophin secretion by this ovarian steroid ceases; oestrogen instead starts promoting hypothalamic LHRH release, as well as augmenting anterior pituitary responsiveness to LHRH. This positive oestrogenic modulation of hypothalamic-pituitary function results in a preovulatory LHRH surge and corresponding surge in LH (Figure 2.2).

2.6 The LH surge, which closely follows the oestrogen peak, occurs during the afternoon of prooestrus and triggers ovulation approximately 10-12 hours later. FSH levels peak twice in the rat; the first (preovulatory) peak is LHRH-dependent and occurs in concert with the LH peak. This is followed by a second (postovulatory) rise in FSH that occurs at the time of ovulation or shortly after. This secondary FSH elevation is thought to be LHRH-independent, reflecting reduced inhibin synthesis by the postovulatory follicle.

The rat corpus luteum is short-lived and regresses in the absence of prolactin stimulation

2.7 Progesterone levels start to increase during prooestrus and peak during ovulation. Like oestrogen, progesterone feedback control of hypothalamic-pituitary function may be negative or positive, depending on the stage in the oestrous cycle (Figure 2.2). Following ovulation, progesterone synergises with oestrogen to inhibit gonadotrophin secretion. Conversely, during prooestrus, rising progesterone levels trigger hypothalamic LHRH secretion, stimulating gonadotrophs in the anterior pituitary and reinforcing the preovulatory LH surge.

2.8 After ovulation, lutenisation of the follicular granulosa and thecal cells occurs resulting in the formation of the corpus luteum. In the rat, the corpus luteum secretes progesterone autonomously for approximately 48 hours, before becoming non-functional and degenerating over the course of several subsequent oestrous cycles.

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2.9 Prolongation of corpus luteum function requires continued pituitary secretion of prolactin, the major luteotrophic hormone in the rat. Prolactin levels peak and fall simultaneously with the preovulatory LH surge in normally cycling rats. Copulation during oestrus causes vaginocervical stimulation which triggers, via a neuroendocrine reflex, twice daily prolactin release by lactotrophs in the adenohypophysis. This mating- induced prolactin secretion disrupts normal oestrous cyclicity by maintaining the newly formed corpus luteum in a functional state, thus prolonging dioestrus.

2.10 The continued secretion of progesterone during this lengthened dioestrus phase initiates development of the endometrium in preparation for implantation of the fertilised ovum, as well as initiating and maintaining vaginal mucification (Table 2.1). If the mating is sterile or implantation fails to occur, the corpus luteum regresses, terminating the prolonged dioestrus phase (referred to as pseudopregnancy) after 12-14 days, thus allowing resumption of normal reproductive cyclicity.

Table 2.1 – Major reproductive hormones in the rat: sources and key functions.

HORMONE SOURCE KEY FUNCTIONS

LUTENISING HORMONE-  Pulsatile secretion of LHRH stimulates gonadotrophs in anterior pituitary RELEASING Hypothalamus  Triggers release of lutenising hormone (LH) and follicle stimulating HORMONE hormone (FSH) (LHRH)

LUTENISING  Stimulates production of androstenedione and testosterone by theca interna Anterior pituitary HORMONE  Follicular granulosa cells utilise androgens in synthesis of oestrogen (adenohypophysis) (LH)  LH surge triggers ovulation

FOLLICLE STIMULATING Anterior pituitary  Stimulates development of follicular granulosa cells HORMONE (adenohypophysis)  Upregulates LH receptor expression (FSH)

 Negative and positive feedback control of hypothalamic-pituitary function  Stimulates: - proliferation and cornification of vaginal epithelium Preovulatory OESTROGEN - increased vascular permeability (Graafian) follicle - neutrophil infiltration of vaginal epithelium and uterine endometrium - proliferation of endometrial stromal fibroblasts and epithelial cells - hypertrophy of uterine myometrium

 Negative and positive feedback control of hypothalamic-pituitary function  Stimulates: Postovulatory - mucification of vaginal epithelium† PROGESTERONE follicle - activation and differentiation of endometrial stromal fibroblasts† (corpus luteum) - endometrial gland secretion - hypertrophy of uterine myometrium

 Vaginocervical stimulation triggers prolactin secretion by lactotrophs Anterior pituitary PROLACTIN  Major luteotrophic factor in the rat – required for maintenance of corpus (adenohypophysis) luteum

Preovulatory INHIBIN  Negative feedback control of pituitary FSH secretion (Graafian) follicle

†requires priming with oestrogen

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Figure 2.2 – Hormonal interactions of the HPO axis. Pulsatile release of lutenising hormone-releasing hormone (LHRH) by the hypothalamus drives pituitary secretion of gonadotrophins. The ovarian oestrogen and progesterone exert feedback control at the level of the hypothalamus and pituitary.

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