Ovarian Dynamics

The ovary performs two major functions, gametogenesis or oogenesis and steroidogenesis or production of steroid hormones. These two activities are started during the foetal life and continued through a dynamic process during various phases of post-natal periods.

The term oogenesis is used to describe the developmental process of the ova (singular ovum) or the female gamete. The oogenesis depends upon the interaction between the oocyte and follicular cells surrounding it. The ovarian follicles are the functional unit of the ovary that appears as a cluster of somatic cells to protect and nourish the germ cell or oocyte. The follicular cells are developed through a process termed folliculogenesis.

22.3.1 Oogenesis

Oogenesis is a complex differentiation process leading to the production of functional oocytes. Oogenesis is initiated at the embryonic stage and completed after puberty. The process of oogenesis is commenced with the migration of PGCs into the gonadal ridge and serves the function of stem cells. It proliferates to form oogonia, remains in ovarian nests and is connected by intercellular cytoplasmic bridges. The somatic cells are the pregranulosa cells, or granulosal cells originated from mesonephros cells or ovarian surface epithelium (species-specific). Oogenesis occurs in the outmost layers of the ovaries and is divided into three different phases. These are oocytogenesis, ootidogenesis, and the maturation phase (oogenesis proper).

22.3.1.1 Oocytogenesis

The primordial germ cells (PGC) of the foetus in the ovary are transformed into the oocyte by a series of mitotic pro­cesses called oocytogenesis, or the prenatal phase of oogen­esis. It has two stages. In the first stage, the PGC is transformed into oogonia (singular oogonium).

Oogonia: Oogonium is a diploid cell that resembles sper­matogonium. An oogonium is generally spherical and larger in shape with a prominent nucleus compared to

Fig. 22.11 Oogenesis. Figure shows the formation of a mature ovum from oogonium. The oogonium is developed into a primary oocyte through mitotic division. The haploid secondary oocyte and a (first) polar body are formed by meiosis-I division. Meiosis-II is continued, and ovulation occurs with the resumption of this cell division, in the presence of spermatozoa (after penetration, during fertilisation) as induced meiosis-II and mature ovum and second polar bodies are formed

the surrounding somatic cells. It can be differentiated microscopically by randomly scattered fibrillar and gran­ular material from the somatic cell with a more condensed nucleus with a darker outline.

Primary oocyte: The oogonium is enlarged and transformed into primary oocytes. This process is completed before birth or immediately after birth. Hence, there will be no scope to develop a primary oocyte after birth. In contrast, spermatogenesis continues after birth and is a transient stage of ova that generates haploid secondary oocytes. The oocytogenesis ends with the development of a pri­mary oocyte.

22.3.1.2 Ootidogenesis

The process of transforming the primary oocytes into ootids by meiotic cell division is called ootidogenesis (Fig. 22.11). The meiotic cell division has two distinct stages, meiosis I and meiosis II.

Meiosis I: It starts during embryonic life but is arrested at the diplotene of prophase I until puberty. After puberty, the cell division is completed under follicle-stimulating hor­mone (FSH). At the end of meiosis I, the synapsis occurs, but each chromosome has still two chromatids, and the primary oocyte is divided asymmetrically into two daugh­ter haploid cells.

Meiosis II: Immediately after meiosis I, the two haploid daughter cells undergo meiosis II. But, for the secondary oocytes, meiosis II is again halted at metaphase II; how­ever, the first polar body completes its meiosis and generates two second polar bodies. The secondary oocyte is called an ootid, and its nucleus is called a germinal vesicle (GV). The ootids are devoid of fertilising capabilities and remain in a dictyate state. This arrest is to inactivate DNA to protect it so that it is not vulnerable to possible damage during its lifetime for proper fertilisation.

22.3.1.2.1 Maturation Phase

The ootids mature into the ovum with fertilising capabilities in the maturation process. The maturation phase is completed before ovulation or immediately after ovulation at the fallopian tube, depending on species (details in Chap. 23). The luteinising hormone (LH) surge controls the meiosis process during ovulation. LH surge facilitates completing the meiosis I and extrusion of the first polar body, followed by halting meiosis II at metaphase II.

22.3.2 Folliculogenesis

The synchronised transformation of ovarian follicles holds the primary oocyte encompasses the growth and development or atresia of follicles through morphological and functional changes is termed folliculogenesis. There are two types of follicular pools within the ovaries: the non-growing pool and the growing pool. The non-growing pool contains primordial follicles, and the growing pool contains primary, secondary, and tertiary follicles. The primordial follicles enter into the growth phase throughout the reproductive life of an animal. The primordial follicle and primary follicle are developed during the prenatal stage. Secondary follicles are found in the antral stage, and the tertiary follicles are formed during the pre-ovulatory stage of follicular development.

22.3.2.1 Ovigerous Cords and Primordial Follicles The pregranulosa cells-oogonia complexes are gradually fused and form a tube-like structure called ovigerous cords. The wall of the cord is made up of pregranulosa cells. The formation of somatic cell-germ cell complexes (pregranulosa cells-oogonia complexes) are mediated through the chemo­tactic attraction between stem cell factor (SCF, or steel fac­tor), a cytokine secreted from the granulosal cells and the expression of the c-kit mRNA/protein receptors (CD 117, a receptor tyrosine kinase) on the oogonial cell surface. The basic fibroblast growth factor (bFGF), released from the oogonia, increases the production of kit ligand in pregranulosa cells and facilitates the interaction. The oogonia that fail to establish this interaction are prone to apoptosis, and the granulosa cells become free to attach with other active oogonia. Ovigerous cords secrets the basal lamina and form the primordial follicle. It is more prominent in rodents, not ruminants, due to long foetal life and slow ovarian development. The ovigerous cords disappear before birth, and the oogonia either transform to the active form (within the primordial follicles) or undergo apoptosis. The primordial follicle consists of an oocyte surrounded by flat­tened (squamous) pregranulosa cells or follicle cells (Fig. 22.13a). These primordial follicles then enter the grow­ing pool and are transformed into primary follicles. The protease enzymes break down the cytoplasmic bridge between the oocytes during follicle formation. Incomplete breakage of cytoplasmic bridges leads to multi-oocyte follicles (MOFs) or polyovular follicles as they contain more than one oocyte.

Fig.

Fig. 22.12 Ovarian cycle. Figure showing the follicular development (clockwise) in two phases, gonadotropin-independent or pre-antral phase and gonadotropin-dependent or antral phase. The follicles from the germ cell pool of the hilum, a part of the ovarian cortex, developed into primordial follicles, followed by primary follicles in the gonadotropin-independent phase. The secondary follicles are devel­oped from primary follicles, followed by antrum follicles under the

influence of follicle-stimulating hormone (FSH). The tertiary follicle or Graafian follicle is developed under the influence of luteinising hor­mone that leads to ovulation with the release of ovum from the rup­tured follicle with subsequent formation of corpus luteum. The ovarian surface also contains the scar tissue bearing corpus albicans of early ovulated follicles. The ovarian cortex also has atretic follicles that fail to form Graafian follicles and don’t undergo ovulation

22.3.2.1.1 FactorsAffecting Primordial Follicle Reserve

The pool of resting primordial follicles is called primordial follicle reserve or Ovarianfollicular reserve. It occurs mainly during 90-140 days of foetal life in cows. The size of follicle reserve is related to the animal’s fertility, and the animal that has more reserve has longer reproductive life. Animals with poor follicular reserve have a weak response to superovula­tion protocol. Some internal and external factors reduce ovarian follicular reserve by altering hormonal steroid levels and reducing the responsiveness of the receptors of these hormones (androgen receptor, AR and estrogen receptor, ER) to the follicular cells. Internal factors such as anti- Mullerian hormone (AMH), growth and differentiation fac­tor 9 (GDF9), bone morphogenetic protein (BMP15), and maternal nutrition and health of dam are reported to alter the primordial follicular reserve.

Serum AMH is undetectable during infancy and rapidly increases with the onset of puberty, reflecting the follicular recruitment. AMH secretion declines when the dominant follicle separates from the antral follicle. The AMH is used as a marker to evaluate ovarian follicular reserve. GFD9 causes growth and differentiation of granulosa cells and expands cumulus cells. The polymorphism of the GDR9 genes is associated with infertility in sheep. Lower expres­sion of GFD9 is reported in women with the polycystic ovarian syndrome (PCOS). BMP 15 causes proliferation and differentiation of granulosa cells and inhibits cumulus cell apoptosis. In ewe, the DEAD (Asp-Glu-Ala-Asp) box polypeptide-4 as a factor with the expression of DDX4, a protein-coding gene, is used as a molecular marker to recog­nise the size of primordial follicle reserve. The level of BMP 15 in the follicular fluid is correlated with oocyte quality. External factors like phytoestrogens and bisphenol A (BPA) or endocrine-disrupting chemicals (EDCs) from plastics, pesticides, and industrial chemicals reduce the follicular reserve.

22.3.2.2 Primary Follicle

The oocyte within the primordial follicle increases its size. The squamous pregranulosa cells are transformed into cuboi- dal and form a layer around the growing oocyte to form primary follicles (Fig. 22.13b). These follicles are generally located under the tunica albuginea at the deepest part of the ovarian cortex. In cows, the primordial follicle and primary follicles are usually formed during 90 and 140 days of foetal life, respectively.

22.3.2.3 Secondary Follicle

The secondary follicle has an oocyte (transformed into oogo­nium), called a secondary oocyte, surrounded by zona pellucida and two or more layers (multilayer) of somatic follicular granulosa cells. These granulosa cells originated from pregranulosa cells. The granulosa cells nearer to the basement membrane are called mural granulosa cells, and the cells closer to the follicular antrum are known as antral granulosa cells. Theca cells are situated over the granulosa cell layer (Fig. 22.14). These follicles are in a growing stage close to the ovarian epithelium. The glycoproteinous substances released from the granulosa cells formed the zona pellucida to cover the oocyte for its protection (Fig. 22.14). The granulosa cells are responsive to FSH and theca cells to LH. The secondary follicles generally start to form during 210 days of foetal life in cattle.

22.3.2.4 Tertiary Follicle or Vesicular Follicle

The secretion of proteoglycans (hyaluronan and chondroitin sulphate) creates an osmotic gradient that drives fluid from thecal vasculature. This fluid (liquor folliculi) accumulates, results in the splitting of granulosa cell layers, and gradually forms a central fluid-filled space called the antrum. In the species with the large follicle, additional alpha-trypsin inhibitors in the follicular fluid cross-link with hyaluronan to facilitate the retention of these molecules within the antrum to maintain the osmotic gradient.

22.3.2.5 Graafian Follicle (Named for the Dutch Anatomist Regnier de Graaf, 1641-73)

Graafian follicles are a heterogeneous family of relatively large follicles with 400 μ (0.4 mm) diameter and 150-200 μ (0.2 mm) in large and small mammals. The cell type of tertiary follicle is similar to the secondary follicle with more granulosa and theca cells and a large antrum; as such, the term antral follicle is synonymous with the Graafian follicle. But, to be more precise, the matured form of antral follicles is called Graafian follicles (Fig. 22.15). FSH and LH trigger the maturation process along with estrogen.

Histologically, the Graafian follicle contains six distinct components, namely the theca externa, theca interna, basal lamina, granulosa cells, oocyte, and follicular fluid. The morphological features of the Graafian follicle do not alter as growth proceeds. Theca externa is the outermost layer characterised by the presence of smooth muscle cells innervated by autonomic nerves. The contractile activity of these smooth muscles facilitates the ovulation process. The theca interna, composed of loose connective tissue and blood vessels, is situated just beneath the theca externa. The thecal cells and granulose cells are separated by a thin layer of extracellular matrix called basal lamina or follicular basement membrane. The granulose cells of the Graafian follicles have four different layers. The layer beneath the basal lamina is called membrana granulosa, composed of a pseudostratified columnar epithelium anchored to the basal lamina. The periantral granulosa cell layer is situated just after the membrana granulosa. The cumulus oophorus (discus proligerus) is a cluster of granulosa cell layers surrounding the oocyte. The innermost layers of the cumulus oophorus adjacent to the zona pellucida are called corona radiate towards the antrum. The cumulus cells are essential for oocyte survival as they provide metabolic and nutritional support to the oocyte. The cumulus cells perform glycolysis and transport pyruvate to the oocytes for energy production as oocytes cannot metabolise glucose independently. The cumulous cells also transport endogenous molecules like ATP, cAMP, and cyclic GMP to maintain meiotic arrest.

Fig. 22.14 Secondary follicle. Figure shows oocyte, zona pellucida, granulosa cells, and theca cells

Fig. 22.15 Tertiary follicle. Figure shows follicle having antrum and oocyte covered with zona pellucida, followed by cumulus oophorus with multilayer granulosa cells and theca interna as well as theca externa

The major functions of the follicular cells are to promote the maturation of the follicle and the oocyte and to release the oocyte from the ovary at the time of ovulation to form the corpus luteum and production of steroid hormones. The other functions of the follicles are to protect the oocyte (building zona pellucida, discussed earlier), meiotic arrest of the oocyte and secretion of yolk-forming materials. The granulosa cells of some hibernating bats can store glycogen to provide the energy to the oocyte.

The first growing follicles are generally available in the innermost part of the cortex, near the medulla, where the proliferating oogonia mainly exist nearer to the ovary’s epi­thelium. Major characteristic differences of various stages of oocyte or premature ovum of mammals and its corresponding follicle can easily be distinguished microscopically (Figs. 22.13, 22.14, and 22.15) with certain features (Table 22.17). The size of oocytes and follicles of different stages are species specific (Table 22.18) and can be identified by ultrasonography.

22.3.2.6 Period of Folliculogenesis

Folliculogenesis is initiated with the commencement of oogenesis and the formation of oogonia. Time taken to start the oogenesis and complete the folliculogenesis is presented in Table 22.19. The primordial follicle reserve is initiated during foetal life, and most of the primordial follicles are formed before birth in cows, ewe, mare, and human. Primor­dial follicle formation is continued after birth in sows, queens, and mice. In the rabbit, both oogenesis and folliculogenesis are initiated after birth.

22.3.2.7 Follicular Waves

The earlier stages of follicular growth (follicles with a diam­eter et al. (2009), Bachler et al. (2014), Conti and Chang (2016), Haque et al. (2016), Hoque et al. (2016), Sasan et al. (2016)

Table 22.19 Period of folliculogenesis in mammals

Source: Monniaux et al. (2014)

growth. The follicular atresia facilitates by hormone- mediated apoptosis of granulosa cells. The ovigerous cords break down during atresia, resulting in apoptosis of pregranulosa cells, followed by replacement of fibrous materials. The oocytes and the oogonia, which are not transformed into the primary oocyte, are degraded and become part of the ovarian stroma. Atresia of the follicle can occur at any stage of development. In cows and humans, rapid follicular atresia is seen at birth, puberty, and certain age of adult life. The last rapid atresia occurs during cows’ 8-10 years of age and 35-40 years in humans. Generally, the follicle number reduces by about 1∕20th between birth and puberty (Table 22.20). The maximum number of follicles is lost before birth in bovines than in other domestic animals.

22.3.2.7.5 Duration of Follicular Wave

The duration of a follicular wave depends upon the selection of dominant follicle(s) and is species specific. Other factors like the stage of lactation, milk yield, nutritional status, postpartum period, and luteal phase duration also influence the duration of the follicular wave. Successive two follicular waves at an interval of 7-10 days are common in cow. Hence,

Table 22.20 Follicular atresia during foetal life in various domestic animals

within a span of one estrous cycle (19-21), 2-3 waves can occur (Fig. 22.16). Similarly, two waves are common in buffalo, 1-2 in the mare and four or more in doe. In humans, inter-wave intervals (IWI) generally vary from 6 to 11 (for 3 wave patterns) and 14 to 15 days (2 wave patterns) in one menstruation cycle (28 days interval).

22.3.2.7.6 Major Wave and Minor Wave

In major waves (ovulatory wave), the divergence of follicles occur, and one follicle becomes dominant and destined for ovulation, whereas others become subordinate. But, no diver­gence occurs in a minor wave (anovulatory follicular wave), and no dominant follicle develops. The loss of follicular

Fig. 22.16 Follicular waves in cattle. Figure shows the three follicular waves in a span of one estrous cycle (21 days) in cattle, comprising one ovulated major wave and two non-ovulated minor waves. The days between two successive ovulations or an estrous cycle period are shown on the ‘X’-axis. The major wave is shown with dark black coloured follicles, and the minor waves are shown with shaded black coloured follicles. Two minor waves originated on day 1 and day 9, respectively, after the day of last ovulation. In contrast, the major wave originated on day 16, when the progesterone level minimises. The events of the follicular wave, separated by horizontal dotted lines, are shown in

divergence in minor waves is due to insufficient gonadotropins and a high progesterone level that restricts the follicular growth below 10 mm in diameter in cattle,

7- 10 mm in sow, 22-23 mm in mare. The major wave is seen in the follicular phase immediately after the luteal phase when the FSH level is more, and the minor wave(s) mainly occurs in the luteal phase. The major and minor follicular waves are well recognised in mares due to their large follicu­lar diameter compared to other species (Table 22.21). In a single estrous cycle, 5-12 follicles have more than 10 mm in diameter, and the largest follicle has a diameter of 35-55 mm. In mares, the major waves are further classified into primary waves in which (one follicle ovulates with oestrus and sec­ondary waves that comprise a dominant follicle that is unable to ovulate or may ovulate after the primary wave by second­ary ovulation. Minor waves are more frequent in spring.

The characteristics of follicular waves in different species have been summarised in Table 22.21.

22.3.3 Ovulation

Ovulation is a biological process in which the oocyte is released from the mature Graafian follicle. It is an inflamma­tory process sequentially controlled by the neuroendocrine boldface, expressing follicular recruitment, followed by selection, growth, and dominance in the form of primary follicles, secondary follicles, antral follicles, tertiary follicles, Graafian follicle. Ovula­tion occurred only from the Graafian follicle of the major wave. The Graafian follicles of the minor waves and all the non-dominated follicles become atretic or regressed. Few primary follicles are involved in the event of recruitment, with the influence of FSH from the primordial follicle reserve or ovarian reserve. Gradual increase of follicle size is represented in the ‘X-axis (not to scale)

Table 22.21 Characteristics of follicular waves in different species

system. Two subsequent events occur at the oocytes and surrounding follicles during the ovulation process. In oocytes, the resumption of meiosis and the structural remodelling of the follicles are happened to release the matur­ing oocyte (Fig. 22.12).

Site of ovulation: The ovulation can occur at any point on the ovarian surface, but the site is restricted to the ovulation fossa in mares. It is probably due to the unique ovarian

Table 22.22 Factors involved in oogenesis, folliculogenesis, and ovulation

Reference: Schuermann et al. (2018)

structure in mares (kidney bean-shaped). The ovulation fossa is situated as a thin, pointed edged surface (wedged-shaped) at the concave side of the ovary. Before ovulation, the large size mature Graafian follicles reach the fossa. In some animals (whales), the ovulation predominates in one ovary; but it alternates between the ovaries in most mammals. The presence of previous corpus luteum restricts the growth of the follicles in rhesus monkeys. So, the ovulation alternates between the ovaries. In the case of cows, sheep, and horses, the ovulation is independent of previous CL’s presence and can occur at random between the ovaries.

22.3.3.1 Theories of Ovulation

There are several theories to explain the mechanism of ovu­lation, and neither of these theories can explain the ovulation process alone.

Follicular pressure theory: During the growth of the follicles, the amount of liquor folliculi increases; this exerts pres­sure on the follicular wall to rupture it.

High osmotic pressure theory: The electrolytes present in the liquor folliculi (Na, K) increase the osmotic pressure, leading to the follicular rupture.

But, these two theories did not accept the same events in cystic ovaries without follicular rupture.

Ischemic theory: The accumulation of follicular fluid exerts pressure on the follicular wall. As a result, ischemia occurs at a point that leads to stigma formation and rupture of the follicle. This theory is partly accepted.

22.3.3.2 Mechanism of Ovulation

A recent theory explains that ovulation is a combination of physiological, biochemical, and biophysical mechanisms triggered by pre-ovulatory LH surge, the involvement of other endocrine and paracrine factors.

The steroidogenesis of pre-ovulatory follicles is regulated through LH receptors present in thecal cells and FSH receptors on granulosa cells. LH stimulates steroidogenic acute regulatory protein (StAR protein) to produce andro­stenedione when it binds with the thecal cell receptors. The androstenedione is diffused into the granulosa cells and converted to estradiol by the action of aromatase (CYP19) under the influence of FSH. Here two cells (theca and granulosa) are involved in estradiol production under the influence of two gonadotropins (LH and FSH). Hence, this theory is called the ‘two cell two gonadotropin theory’ (Fig. 22.3). There is more estradiol production in the late follicular phase, and it achieved its highest critical value. There is a dramatic change in the action of estradiol from negative to positive feedback for the secretion of GnRH and LH, respectively, at the pituitary and hypothalamic levels. As a result, the LH-secreting cells of the anterior pituitary become highly sensitive to GnRH. LH surge controls several key events of ovulation, such as the resumption of oocyte meiosis and expansion of the cumulus-oocyte complex, and follicular remodelling.

22.3.3.2.1 Induction of the Resumption of Oocyte Meiotic Maturation

The mitotic arrest in the oocyte is maintained by a high concentration of cyclic adenosine monophosphate (cAMP). Adenylyl cyclase 3 (AC3) helps in cAMP synthesis, whereas oocyte-specific phosphodiesterase (PDE3A) causes cAMP breakdown. The steady level of high cAMP throughout mitotic arrest is maintained after the inhibition of PDE3A with the production of cyclic guanosine monophosphate (cGMP) by NPR2 guanylyl cyclase of granulose cells (mural and cumulus). A high level of LH at the time of ovulation reduces the intra-oocyte cAMP level by down­regulating NPR2 guanylyl cyclase. The high level of LH also minimises the gap junctions between the oocyte and CGCs and facilitates the diffusion of cGMP within the follicles. The reduced cAMP levels within the oocytes further activate the maturation promoting factor (MPF), a kinase that helps germinal vesicle breakdown (GVBD). The germinal vesicle (GV) is the spherical nucleus of the oocyte, which contains chromatin (DNA) and the nucleolus. GVBD refers to the dissolution of the oocyte nucleus. The MPF also favours the spindle assembly and chromosome segregation to complete the first meiotic division and the formation of the first polar body. The diameter of both follicle and oocyte governs the initiation of meiotic resumption. The antral folli­cle and oocyte size are essential parameters to evaluate oocyte in vitro maturation (IVM) in Assisted Reproductive Technology (ART). In cow, ewe and doe, resumption of meiosis occurs when the diameter of the oocyte, including zona, is generally more than 110 μm as well as the follicle reaches 2-3 mm in diameter. But, follicles having more than 5 mm in diameter of these species are shown to perform better in embryo production. However, in mare, COC com­pactness is considered a better criterion than the diameter of oocyte and follicle.

22.3.3.2.2 Cumulus Oocyte Complex (COC) Expansion Pre-ovulatory LH surge induces the expression of epidermal growth factor-like ligands (EGF-Ls), such as amphiregulin, epiregulin, and betacellulin, and several transcription factors like CCAAT enhancer-binding protein (C/EBP) αZβ and pro­gesterone receptor (PGR). EGF-Ls also stimulate the produc­tion of pentraxin 3 (PTX3) and hyaluronan synthase 2 (HAS2). These are the cumulus matrix proteins that cause the expansion of COC.

22.3.3.2.3 Follicular Remodelling and Rupture

LH surge causes an increase in PGF2α and PGE2 levels. PGE2 stimulates the production of plasminogen activators and increases plasminogen activity, enhancing the follicular wall elasticity and cell migration for mixing theca and granulosa cells during the formation of CL. PGF2α causes the rupture of epithelial cell lysosome at the follicular epithelium. The hydrolase’s from the lysosome cause hydro­lysis of albuginea cells and theca cells. Combining these enzymes and cellular apoptosis leads to thinning extracellular matrix (ECM) layers after hydrolysing laminin, collagen type-IV, fibronectin, and proteoglycans. Thecal fibroblasts are migrated, the surface epithelial cells are sloughed from the follicle at the apex region, and a thin and a circumscribed area is formed called stigma. Rapid dissolution of these ECM components results in apical puncture and channel for the COC. This perforation makes a passage for the vascular constituents during the formation of the corpus luteum imme­diately after ovulation. PGF2α causes contraction of smooth muscles of ovarian stroma and theca externa leading to ovar­ian and follicular contractions. Ovarian contractions lead to follicular rupture, and follicular contractions lead to the expulsion of the oocyte. After the eviction, the ovum and surrounding cells in a gelatinous mass protrude at the ovarian surface and are swept into the ostium by mobile kinocilia of the fimbriae.

In some cows, acute inflammatory reactions occurred during collagenolysis and the release of histamine and prostaglandins with leukocyte recruitment. Under this cir­cumstance, the follicle becomes luteinisation without ovula­tion and forms a cyst (cystic corpora lutea). To hasten this inflammatory process, some immunological factors, like Intercellular Adhesion Molecule 1 (ICAM-1), are secreted in response to LH surge to recruit leukocytes, mainly macrophages and neutrophils in follicular and perifollicular thecal layers, respectively.

22.3.3.3 Types of Ovulation

Ovulation is of two types based on the underlying neuroen­docrine mechanisms. In spontaneous ovulators, like cattle, sheep, goats, pigs, horses, rats, mice, monkeys, and humans, the LH surge is induced by Graafian follicles’ ovarian steroids (estradiol). In contrast, the induced ovulator, like rabbits, ferrets, cats, and camels, mating is the main stimulus to cause LH surge instead of ‘spontaneous’ steroid-induced LH. The somatosensory stimuli originate from the sensory neurones of the female genital tract of these species and activate the noradrenergic neurons of the midbrain and brainstem. These noradrenergic neurons have their projections in the MBH and promote GnRH release, which in turn causes a pre-ovulatory LH surge. A goat is a sponta­neous ovulator, but the presence of a male in the flock of goats (male effect) induces an increase in the frequency of LH that gives rise to a pre-ovulatory LH surge. The evolu­tionary origin of induced ovulation can well explain through male-induced activation of the GnRH neuronal system in females of several species.

A single oocyte is released during ovulation in monotocous species like cows and mares. In polytocous animals, like pigs, dogs, cats, and rats, more than one oocyte is released from different Graafian follicles during ovulation. The small breeds of bitch can ovulate 2-10 oocytes, and the large breed can 5-20 oocytes in one ovulation process. The ovulation can occur from both ovaries at a time in polytocous animals, whereas a single ovary is involved in monotocous animals.

In rare conditions, when two growing follicles reach nearly 10 mm in size with similar sensitivity to FSH, they can transform into two simultaneous dominant follicles. It causes the ovulation of two Graafian follicles at a time and leads to dizygotic or non-identical twins. In superovulation protocol, more numbers of growing follicles become equally responsive to FSH, causing ovulation of many follicles in each wave.

22.3.4 Control of Oogenesis, Folliculogenesis, and Ovulation

22.3.4.1 Endocrine Control

The early stages of follicular development are gonadotropin­independent, as the single layer of cuboidal granulosa cells is non-responsive to gonadotropins. FSH and LH essentially require antrum formation and follicular growth beyond 9 mm diameter. The granulosa cells of secondary follicles are responsive to FSH. High levels of FSH, low LH, and no inhibin initiate follicular recruitment. The gonadotropin stimulates mitosis of granulosa cells and antral fluid forma­tion. FSH also increases the sensitivity of granulosa cells for LH by increasing LH receptors. LH receptors only stimulate the thecal cells from the beginning of thecal cell formation.

When the follicles enter the selection stage, inhibin is produced from the follicles and inhibits FSH release. At the stage of follicular dominance, the larger follicles produce more estrogen and inhibin. A higher estrogen level stimulates a pre-ovulatory LH surge. Inhibin reduces FSH secretion and causes the antral follicles to undergo atresia. Insulin-like growth factors (IGFs) are also involved in follicular growth. IGF stimulates the PI3K pathway that mediates primordial follicle activation. They also sensitise the granulosa cells for FSH action during the terminal phase of follicular develop­ment. The actions of IGFs are controlled by a series of IGF-binding proteins (IGFBP), namely IGFBP-2 and IGFBP-4, secreted from blood or synthesised locally within the follicles. The decreased concentration of IGFBP-2 and IGFBP-4 during the follicular growth leads to increased bioavailability of IGF. Therefore, the low amount of IGFBP4 facilitates dominant follicles to attain FSH indepen­dence, whereas higher levels of IGFBP2 in subordinate follicles lead to follicular atresia. The follicular cells synthesised steroid hormones that regulate the follicular mat­uration processes. Larger follicles produce more estrogen.

The corpus luteum produced after ovulation produces pro­gesterone to maintain pregnancy.

Follistatin synthesised from granulosa cells competes with activin for its receptor; thus, it counteracts the activin. Follistatin inhibits folliculogenesis by inhibiting bone mor­phogenetic protein-15 (BMP-15), a stimulatory factor for granulosa cell proliferation. Recently, an intraovarian factor, C-type natriuretic peptide (CNP), has been involved in the preantral and antral follicle growth and the inhibition of oocyte maturation. CNP is secreted from the granulosa cells in response to FSH and acts to its receptor NPRB expressed in the secondary follicles to stimulate follicular development through cGMP production. But, in the cumulus cells, CNP inhibited phosphodiesterase 3A (PDE3A) enzyme and increased intra-oocyte cAMP levels, suppressing oocyte mat­uration. The CNP level decreases in response to pre-ovulatory LH surge to complete meiosis within pre-ovulatory oocytes. In human, α-fetoprotein, a steroid- binding protein, blocks estrogens and progesterone in the ovary; thus, it controls the folliculogenesis process.

The ovulation is induced by an LH surge (generated by a higher estradiol level) that triggers a biochemical cascade. PGE2 causes the activation of tPA and uPA to augment follicular remodelling along with PGF2α that causes the rupture of epithelial cell lysosome at the follicular epithelium. LH surge also upregulates progesterone receptors in the granulosa layer of bovine pre-ovulatory follicles. The proges­terone stimulates collagenase activity and helps in follicular wall thinning. PGF2α induces ovarian and follicular contractions before ovulation. The GnRH pulse happens to be very rapid with a constantly higher level throughout the LH surge. But this constant exposure leads to desensitisation of LH secretion, resulting in termination of LH surge. Imme­diately after the ovulation, the ruptured follicles are transformed into corpus luteum and release progesterone. In the absence of conception, the corpus luteum regressed leads to decreased progesterone and inhibin. It favours pulsatile GnRH secretion and FSH to promote the growth of the new follicles.

22.3.4.2 Molecular Control

There are nearly 70 genes involved in folliculogenesis in mammals. Several factors and hormones control them. The primordial follicle activation is mediated through phosphoinositide 3-kinase/phosphatase and tensin homolog deleted on chromosome 10 (PI3K/PTEN) pathway regulating cell proliferation and apoptosis. The activation of the PI3K pathway leads to the activation of AKT, a protein kinase that mediates cell proliferation and survival by regulating the phosphorylation of transcription factor forkhead box O-3 (FoxO3). Usually, this FoxO3 induces the transcription of cell cycle arrest genes. It becomes phosphorylated, translocates from the nucleus to the cytoplasm, and is inactivated to allow follicular development. PTEN is a nega­tive regulator of PI3K pathway. The arrest of primordial follicles is controlled through a separate signalling pathway involving tumour suppressor tuberous sclerosis complex 1 (TSC1) and the mammalian target of rapamycin complex 1 (mTORC1). Tsc1 negatively regulates mTORC1 to main­tain follicular quiescence. The activation of AKT also phosphorylates and inactivates Tsc1 to begin follicular awak­ening. Genes that regulate the meiotic arrest of oocytes are species specific. In cows, the YBX2 gene is involved in meiotic arrest. The gene is expressed around 140 days of foetal life and can be used as a marker for the diplotene oocytes. The meiotic arrest is triggered by cyclic GMP, which diffuses into the primary oocyte through the gap junctions and stimulates the phosphodiesterase (PDE3) to promote the degradation of cyclic AMP and subsequent meiotic arrest.

Apoptosis of the germ cells (oogonia) occurs due to lower cKIT gene expression in germ cells. The atresia of the follicles is mediated through time-dependent changes in pro-apoptotic factors and anti-apoptotic factors that regulate cell death. Lower expression of anti-apoptotic B-cell lymphoma/leukaemia-2 (Bcl-2) family of proteins (helps in cell survival) and higher expression of pro-apoptotic BAX trigger follicular atresia. CASP2, released from both germ cells and granulosa cells, regulates apoptosis. Several other factors in oogenesis, folliculogenesis, and ovulation are summarised in Table 22.22.

22.3.4.3 Oocyte-Derived Paracrine Factors

The oocyte secreting different paracrine factors regulates the differentiation of somatic granulosa cells and reproductive hormones (Fig. 22.17). R-spondin-2, growth differentiation factor-9 (GDF9), and bone morphogenetic protein-15 (BMP15) are the three important paracrine factors that regu­late granulosa cells’ growth and differentiation. These three factors are transforming growth factor β (TGF-β) superfamily proteins. The R-spondin-2 transcripts are identified only in the oocytes of primary and larger follicles. R-spondin-

2 promotes the development of primary follicles to the sec­ondary stage and functions like FSH. GDF9 promotes the growth of follicles beyond the primary stage. BMP15 is involved in follicular development. The mutation of bone morphogenetic protein (BMP15, in mouse and human) and growth differentiation factor (GDF9, in mouse and human) genes reduce the protease activity and induce multi-oocyte follicles (MOFs) formation.

22.3.4.3.1 Postpartum Ovulation

IGF-I and insulin promote estradiol production. In high- yielding dairy cows, its role has significantly been identified in resuming ovarian function and ovulation in postpartum conditions. The role of IGF-I signifies ‘metabolic signals’ for ovulation. Poor nutritional status like lesser body fat, glucose, non-esterified fatty acids (NEFA), total cholesterol and aspar­tate aminotransferase (AST), as well as low body condition score (BCS) with high GH, is the primary reason for the anovulatory condition in postpartum state. In cows, at least

3 weeks postpartum is required for ovulation. However, after 5 days of calving, the medium-sized follicles may grow into large follicles but cannot ovulate instead of becoming atretic.

22.3.5 Corpus Luteum

The temporary heterogeneous endocrine structure consists of steroidogenic luteal cells, fibroblasts, endothelial, pericytes, and immune cells formed in the ovum-free follicle on the ovarian surface called corpus luteum (in plural corpora lutea).

Fig. 22.17 Role of paracrine and endocrine factors in folliculogenesis. In the primordial follicle reserve, some primordial follicles are in a dormant state induced by dormancy factors such as TGF-β and E-cadherin. Some of the primary follicles are activated by mTOR and AKT signalling and give rise to the primary follicle. Insulin-like growth factor-I (IGF-I) also promotes the growth of primordial follicles.

Follicle-stimulating hormone (FSH) and oocyte-derived paracrine factors like growth differentiation factor 9 (GDF9), bone morphoge­netic protein (BMP6, BMP15), and fibroblast growth factors (FGFs) regulate the growth and differentiation of primary and secondary follicles. FSH induces aromatase expression for estrogen biosynthesis, and a high estrogen level stimulates LH surge for ovulation

Table 22.23 Cellular constituents of corpus luteum and their endocrine products

It plays a central role in regulating the reproductive cycle and maintenance of pregnancy. The process of formation of corpora lutea is known as luteinisation. During luteinisation, the granulosa and theca cells transform into lutein cells that can produce significant amounts of progesterone and moder­ate estradiol and inhibin A (Table 22.23). In Latin, ‘lutin’ means yellow. The cow’s corpus luteum (CL) is yellow due to the large quantity of lutein pigment (carotenoids). But, it is mostly red in other species due to high Vascularisation. The cells of CL have distinguished morphological, endocrine, and biochemical features that enable them to secret a wide range of endocrine products (Table 22.24).

22.3.5.1 Formation of CL

The formation of the CL begins with the expression of genes to regulate cell cycles in theca and granulosa cells, followed by the breakdown of the follicular basal membrane to allow the migration of endothelial cells, fibroblasts, and theca cells into the avascular granulosa layer. Many tiny blood vessels rupture at ovulation, leading to haemorrhage, and it appears as a blood clot penetrates at the centre of the former follicle. It is called corpus haemorrhagicum. Locally released PGE2 activates the plasmin to absorb the clotted materials and induce cellular differentiation. Corpus haemorrhagicum sub­sequently becomes functional CL by tissue remodelling and vascularisation.

22.3.5.1.1 Upregulation of Genes for Luteinisation

The binding of LH with its receptors activates the signalling pathway through the stimulatory guanine nucleotide-binding protein Gs and adenylyl cyclase (AC) to increase cAMP and activate cAMP-dependent protein kinase (PKA). Upon acti­vation, the catalytic unit of PKA translocates to the nucleus and phosphorylates several transcription factors required for luteinisation. They are mitogen-activated protein kinase3 (MAPK3), matrix metalloproteinases (MMPs), tissue inhibi­tor of metalloproteinases (TIMP), JunD, Frizzled Class Receptor 4 (FZD4), estrogen receptor (ERa/ERp), prolactin receptor (PRL-R), steroidogenic acute regulatory protein (stAR), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endocrine gland-derived VEGF (EGVEGF), cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD), and FSH receptor (FSH-R).

22.3.5.1.2 Tissue Remodelling

LH-induced expression of several MMPs and TIMPs facilitates tissue remodelling by disrupting the extracellular matrix (ECM), followed by cell migration and neovascularisation. MMP-2 (gelatinase A), expressed in luteal and endothelial cells, and MMP-9 (gelatinase B), expressed in stromal cells, are two important MMPs involved in tissue remodelling by cleaving type IV collagen. Alpha 2 macroglobulin (α2M) and TIMPs inhibit MMPs. Prolactin stimulates the expression of α2 macroglobulin. The ECM acts as a ‘scaffold’ protein to hold the luteal cells. The theca and granulosa cells undergo extensive hypertrophy and dif­ferentiate into steroidogenic luteal cells. Granulosa cells are differentiated into large luteal cells, and theca cells are transformed into small luteal cells. Large luteal cells synthesised 2-40 folds more progesterone than small cells in ruminants and rodents. In several species, a considerable mixing between these cell types occurred during the luteinisation process, except in primates, where two cell populations remain relatively separate and designated as granulosa-lutein cells and theca-lutein cells, respectively. At the initial stage of luteinisation, all the lutein cells proliferate and contain large quantities of fat droplets (cholesterol as the precursor for progesterone) within their cytoplasm. The structure gradually becomes a solid mass over the surface of the ovary. Later, the cells become hypertrophied, and the vascular network regenerates throughout the structure with the presence of fibroblast. The large lutein cells have abun­dant rough endoplasmic reticulum, prominent Golgi bodies, and secretory granules, making them endocrine cells and synthesising progesterone.

Table 22.24 Functional characteristics of corpus luteum (CL) in different species

Source: Compiled from various sources

LH luteinising hormone, PRL prolactin hormone, hCG human chorionic gonadotropin, E2 estrogen, PGE2 prostaglandin E2, Pri primary, PGF2α prostaglandin F2α, Preg pregnancy

22.3.5.1.3 Vascularisation

Neovascularisation is essential for the formation and mainte­nance of CL as it requires profuse blood flow to transport nutrients, hormones, and lipoprotein-bound cholesterol. Immediately after ovulation and LH surge, theca-derived pericytes (the first vascular cell) start invading luteal paren­chyma and proliferates rapidly to give rise to many blood vessels in the CL. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and endo­crine gland-derived VEGF (EGVEGF) help in CL angiogen­esis. These growth factors are expressed in response to LH.

The formation of the corpus luteum takes 8-9 days in cow and sow. In non-pregnant animals, the functional CL regresses after 17-18 days and completely regressed within 19 days. Upon regression, it becomes an avascular scar tissue called corpus albicans. The functional CL persists throughout the gestation period in pregnancy and is called the corpus luteum graviditatis. LH controls the development and main­tenance of CL function. In pregnant women, the functionality of CL is also maintained by the hormones like human chori­onic gonadotropin (hCG, like LH) secreted from a blastocyst (trophoblast) starts at day 9 post-fertilisation.

22.3.5.2 Functions of CL

22.3.5.2.1 Luteal Steroidogenesis

The expression of key proteins involved in the uptake, trans­port, and processing of cholesterol to progesterone enables the corpus luteum to act as a transient endocrine organ during and after luteinisation. The large and small luteal cells con­tribute around 85% and 15% progesterone synthesis in the cow. The progesterone secreted from CL helps in the embryo’s implantation and maintenance of the pregnancy. The luteal steroidogenesis begins with the uptake of choles­terol. CL can incorporate cholesterol from both HDL and LDL. Still, HDL is the main source of cholesterol for the CL due to scavenger receptor class B type I (SR-BI) and HDL receptor for selective uptake of HDL-derived cholesterol ester. The expression of SR-BI is increased several folds in response to LH/hCG during the development of the CL. But, due to its hydrophobic nature, cholesterol requires a carrier protein named sterol carrier protein-2 (SCP-2) for its intra­cellular movement to the mitochondria where the steroido­genic enzymes are located. The mitochondrial P450scc transforms cholesterol into progesterone. But the amount of progesterone secretion not only depends on the amount of cholesterol or the expression of P450scc. Another enzyme, 20α-HSD, that catabolises progesterone into the inactive 20α-DHP, also controls the progesterone secretion. PRL, LH, and estradiol regulate the proteins involved in luteal steroidogenesis.

In humans and rodents, CL also synthesises androgens and estrogens in addition to progesterone. The androgen produced from the CL is androstenedione. The enzyme P45017 a-hydroxylase/C17-20 lyase (P450c17 or CYP17) helps converts progesterone into androstenedione. But, in pre-ovulatory follicles, P450c17 is expressed only in theca and interstitial cells, not in granulosa cells.

22.3.5.2.2 Synthesis of Protein Hormones

The CL also produces relaxin responsible for softening the pubic symphysis and uterine cervix and the relaxation of the myometrium during parturition. Large luteal cells produce oxytocin, which helps synthesise prostaglandin from the uterus in ruminants and pigs.

22.3.5.3 The Fate of CL

The corpus luteum has two fates depending on the occurrence of fertilisation. If fertilisation and implantation occur, the CL persists throughout the pregnancy to secret progesterone and maintains the pregnancy as corpus luteum graviditatis. The CL is regressed to form corpus albicans if fertilisation doesn’t occur in the alternate fate. The life span of the corpus luteum is governed by the synchronised activity of the pituitary, ovary, uterus, and the embryo through LH, progesterone, oxytocin, and prostaglandin. The substances that support CL to sustain are called luteotropic, and those that terminate the CL are termed luteolytic. The actions of luteotropic and luteolytic substances on CL are different in various species. The LH, progesterone, and prostaglandin E2 (PGE2) are the luteotropic, whereas oxytocin and prostaglandin F2α (PGF2α) are luteolytic in most of the domestic species. In rodents and carnivores, prolactin plays a vital role in CL formation. In many, but not all, animal species, luteolysis is mediated by uterine prostaglandin F2α (PGF2α); hence, called primary luteolysin. Luteolysis occurs through the induction of multiple biochemical pathways that inhibit pro­gesterone secretion and apoptosis. Uterine PGF2α is the major substance that causes luteolysis in ruminants, pigs, horses, guinea pigs, hamsters, rabbits, and rats. Placental PGF2α is the major luteolytic substance in canine or feline species (discussed later). Estrogens are considered luteotro- pic in some species like pigs, rats, and rabbits. LH also helps to sustain the life of CL. During the persistence of CL, no FSH is synthesised from the pituitary due to the high secre­tion of progesterone from the CL. But, immediate after regression of CL, the FSH can be synthesised in the pituitary, and a new follicular wave or a new cycle can start. Hence, luteolysis is the key factor in initiating the cycle for ovulation of follicles, and synthetic luteolytic drugs are administered to augment fertility and estrus synchronisation and the super­ovulation process.

22.3.5.3.1 Synthesis of PGF2α

During the middle of the diestrus, prolonged exposure to progesterone down-regulates its receptor at the uterine endo­metrium, leading to higher estrogen receptor expression. It generally occurs around day 14 of the estrous cycle in cattle. After binding with its receptor, the estrogens upregulate the oxytocin receptor at the endometrium. The oxytocin facilitates the conversion of arachidonic acid to prostaglandins (PGF2α) (Fig. 22.18). Pulsatile secretion of PGF2α causes luteolysis. The pulsatile release of PGF2α and the appearance of the local luteolytic factors are species specific (Table 22.7). It takes 18-19 in cow, 14-15 in mare, 13-16 in sow, and 11-12 days in women unable to conceive.

In pregnant animals, the IFN-τ released from the concep­tus suppresses estrogen receptor expression at endometrium vis-a-vis synthesis of PGF2α to sustain the CL. Thus, foetus acts as a luteotropic substance. In the case of polytocous species, the number of foetuses determines the PGF2α syn­thesis. In pigs, less than four foetuses may cause abortion due to luteolysis with the action of PGF2α. A high progesterone level can also inhibit the synthesis of oxytocin and PGF2α.

Fig. 22.18 Biosynthesis and metabolism of Prostaglandin F2α (PGF2α). Oxytocin activates cytoplasmic Phospholipase A2 (cPLA2) and facilitates its translocation from cytoplasm to the membrane of endoplasmic reticulum and nucleus where the activated PLA2 converts membrane glycerophospholipids (Phospholipids) into Arachidonic acid in the presence of calcium ion (Ca²+). Arachidonic acid is converted to Prostaglandin F2α (PGF2α) by the actions of Cox1, Cox2, and prostaglandin (PG) F Synthase with the intermediate

products like Prostaglandin G2 (PGG2) and Prostaglandin H2 (PGH2). PGH2 can be transformed into Prostaglandin D2 by Prosta­glandin (PG) D Synthase. Similarly, Prostaglandin E2, Prostaglandin I2, and Thromboxane A2 can also be synthesised by their synthases. The PGF2α is metabolised to its inactivated forms like 15-keto-13,

14- dihydro PGF2α (PGFM), under the influence of Prostaglandin

15- dehydrogenase (PGDH)

Thus, small-sized CL is regressed earlier due to its low progesterone secretion.

Due to its short biological half-life, PGF2α cannot be measured directly in serum. Instead, its plasma metabolites 15-keto-13,14-dihydro-PGF2α (PGFM) can be estimated and obtain an indirect measurement of PGF2α.

22.3.5.3.2 Transport of Uterine PGF2α to the Ovary

The PGF2α is synthesised and secreted from the endome­trium in a pulsatile manner and directly reaches the ovary through a unique vascular utero-ovarian plexus (UOP), bypassing the systemic circulation. Uterine PGF2α enters the uterine vein, and the uterine vein forms the utero-ovarian vein after joining the ovarian vein. From this utero-ovarian vein, PGF2α enters the ovarian artery through the utero- ovarian plexus (UOP) and ultimately reaches the ovary for luteolysis. In ruminants, local synthesis of PGF2α occurs through an autocrine-signalling loop. The uterine PGF2α upregulates the expression of prostaglandin-endoperoxide synthase 2 (PGHS-2) or cyclooxygenase-2 (COX-2) in luteal cells to synthesise PGF2α locally.

22.3.5.4 The Regression of CL (Luteolysis)

The regression of the CL occurs in two phases, functional regression associated with a decrease in progesterone synthe­sis and structural regression, where the luteal cells undergo programmed cell death. The structural regression initiates after the initial decline in progesterone. In rats and some other species, the CL persists even after functional luteolysis and produces some inactive metabolites of progesterone. In most domestic species, both types of luteolysis coincide. In cows, the concentration of progesterone below1 ng/mL denotes functional luteolysis.

22.3.5.4.1 The Role of PGF2α on Functional

Regression

PGF2α doesn’t inhibit progesterone synthesis, but it causes the metabolism of progesterone to 20-αDHP through the activation of the enzyme 20-α-hydroxysteroid dehydroge­nase (20αHSD). In addition to this, PGF2α is also shown to reduce cholesterol transport by decreasing SCP-2 and StAR expression. PGF2α blocks LH-induced cAMP accumulation and thus mediates anti-LH action on luteal cells. Several other transcription factors are reported to be involved in functional luteolysis, such as nur77 and endothelial cell- derived peptide endothelin-1 (ET-1). Transcription factor nur77 induces 20αHSD, whereas ET-1 directly inhibits pro­gesterone secretion.

22.3.5.4.2 The Role of PGF2α on Structural

Regression

PGF2α are involved in several cellular and molecular events that lead to structural luteolysis, such as vasoconstriction, apoptosis, infiltration of immune cells, increased metalloproteinase activity, and induction of oxidative stress.

22.3.5.4.2.1 Vasoconstriction

PGF2α induces the expression of several vasoactive factors, such as endothelin-1 (ET-1), endothelin converting enzyme (ECE), endothelin type A and B receptors (ETA-R/ETB-R), angiotensin II (Ang II) and angiotensin-converting enzyme (ACE). During the initial stage of luteolysis, there is increased blood flow to the CL through nitric oxide (NO) production. PGF2α stimulates this NO production by activating endothelial nitric oxide synthase (eNOS). Due to increased blood flow and secrets ET-1 and Ang II, the capillaries are subjected to high shear stress. These two local factors induce chronic vasoconstriction of the CL arterioles.

22.3.5.4.2.2 Apoptosis

PGF2α, along with other factors like PRL, TNF, and Fas ligand, triggers cell death signals. The apoptosis is mediated by both the extrinsic or death receptor-mediated pathway and the intrinsic or extrinsic mitochondrial pathway through the activation of caspases. In the extrinsic pathway, the Fas ligand (FasL), after binding with the TNF receptor (TNFR- 3, -4, and -5), activates caspase-8. On the other hand, the intrinsic pathway is activated in response to stress stimuli that alter the mitochondrial membrane permeability to release cytochrome c. The cytochrome c, in turn, combines with protease-activating factor-1 and procaspase-9 to form an apoptosome that ultimately activates caspase-9. These caspases, in turn, cleave some essential intracellular peptides like actin, poly (ADP-ribose), polymerase (parp) (involved in DNA repair), and protein kinases to facilitate cell death.

22.3.5.4.2.3 Immune Cell Infiltration

PGF2α induces the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) in the luteal cells, resulting in monocyte/ macrophage infiltration into the CL and phagocytose the luteal cells.

22.3.5.4.2.4 Increased Metalloproteinase Activity

The expression of luteal cell-specific tissue inhibitor of metalloproteinase-1 (TIMP-1) is induced by PGF2α. TIMP- 1 increases the metalloproteinase activity to cause structural regression.

22.3.5.4.2.5 Induction of Oxidative Stress

PGF2α decreases the activity of the antioxidant machinery of the CL by reducing protective enzymes and antioxidant vitamins (ascorbic acid). It also causes the down-regulation of genes necessary to eliminate free radicals.

22.3.5.5 Persistent CL

Persistent CL is a pathological condition where the CL is failed to regress beyond the day of structural Iuteolysis (day 19 in cow and day 16 in mare). It may be due to delayed ovulation, embryonic loss after the maternal recognition of pregnancy, and chronic uterine infections. Within 4-5 days post ovulation, uterine infections damage the receptors for progesterone and oxytocin in the endometrium, interfering with PGF2α synthesis and persistent CL. The administration of nonsteroidal anti-inflammatory drugs interferes with the endometrial PGF2α synthesis and may cause persistent CL. In persistent CL, growing follicles of the existing follic­ular waves become dominant; but fail to ovulate. The animals are not in heat and can be mistakenly considered pregnant. There are some distinguished features of persistent CL in contrast to cyclic CL and CL during pregnancy. In mare, persistent corpus luteum appears as a well-demarcated hyperechoic structure on the ovary, with better uterine tone and pale, tight, and dry cervix examined on speculum. The expressions of immune tolerance-related factors (PGES and forkhead/winged-helix transcription factor 3) were upregulated in pregnancy CL but not in persistent CL.

The upregulation of genes involved in lymphangiogenesis, inflammation, and apoptosis were in the same proportion in persistent and CL of pregnancy, but not in the case of cyclic CL. The diagnosis of persistent CL can be made upon history, rectal palpation, progesterone assay, a transvaginal ultrasound, USG-guided ovarian, and endometrial biopsy. The level of serum progesterone concen­tration higher than 1.0 ng/mL is reported in mare exhibiting persistent CL. The treatment protocol for persistent CL in cows includes the injection of PGF2α alone or in combina­tion with GnRH (48-56 h after PGF2α injection). However, a single PGF2α injection is reported to give better results in the treatment of persistent CL. Sometimes, uterine wash or mes­saging stimulates the synthesis of the PGF2α and is applied to induce luteolysis. Some contraceptives, like the intrauterine device (IUD), are used to diminish the PGF2α synthesis from the endometrium and induce transient sterility in animals.

Know More.............

Longest CL

The CL with the most extended life span among mammals is found in the lynx (animal under the Felidae family). Physiologically persistent corpora lutea exist after pregnancy and pseudo-pregnancy for up to 2 years and can secret progesterone. Thus, folliculogenesis is inhibited during the non-breeding season(s). The progesterone level is temporarily reduced during the period of estrus and parturition in the breeding season and induces this polyestrous ani­mal as a monoestrous. The presence of a potential higher level of superoxide anions causing apoptosis of the growing follicles has been identified in lynx and is a physiological adaptation.

22.3.5.6 CL in Dogs and Cats

The corpus luteum of bitch remains functional for a particular period, whether the bitch is pregnant or not. The secretion of progesterone (P4) is similar in pregnant and non-pregnant bitches throughout the entire luteal life span before the prepartum luteolysis. The luteal phage ends with the onset of parturition (around 65 days) in pregnant bitches; but in non-pregnant bitches functional CL exists for a more extended period (75-100 days). The major luteotropic factors in bitches are PGE2 (initial phase) and prolactin (later phase, 25 days from the CL formation) in synergy with LH. The prostaglandin E2 (PGE2) as a luteotropic factor (at the initial luteal phase) is identified that acting through an autocrine/ paracrine fashion. PGE2 is mediated by the upregulation of steroidogenic acute regulatory (StAR) protein. The prolactin acts as a luteotropic factor from 25 days of the luteal phase. There are several other factors, like lymphocytes (CD4 and CD8), cytokines (IL8, IL10, IL12, TNFα, or TGFβ), tropic factors (IGF and VEGF), and some glucose transporters and insulin-sensing receptors also act as luteotropic factors in these species. Another uniqueness of canine CL is that the uterus has no role in CL regression. It is probably due to anatomic independence between the uterine vein and the ovarian artery. Progesterone and estrogen also have a luteo- tropic role. The ageing of CL brings about the regression of CL. A passive degenerative process in association with apo- ptotic signals is evident during the time of luteolysis in ferret and mink due to reduced expression of PRLR and LHR in CL. However, the exogenous PGF2α can induce luteolysis in non-pregnant (after day 30 of the luteal phase) and pregnant (in the second half of pregnancy). In queen, the CL may continue a maximum of 40-50 days in the non-pregnant luteal phase (also called pseudo-pregnancy) or approximately 65 days in pregnancy. In dogs, the CL is the only source of progesterone, unlike in cats, where placental progesterone can support the pregnancy from day 40 to 45 of gestation. Diminished progesterone levels from day 45 of pregnancy induce utero/placental PGF2α release, which stimulates StAR expression and the appearance of vacuoles and connec­tive tissue elements, collagen fibres, and apoptotic factors in luteal cells. These factors lead to luteolysis in pregnant bitch during the time of parturition. In the case of induced ovulator like cats, rats, and rabbits, pseudo-pregnancy is developed in non-fertilised ovulation.

22.3.5.7 CL in Mares

In mares, two corpora lutea form during gestation. The pri­mary is formed due to ovulation and exists up to the 35th day of pregnancy. It is the primary source of progesterone during this period. The primary CL is regressed around 25-30 days of gestation, and the secondary CL is formed as a result of ovulation or luteinisation of follicles under the influence of equine chorionic gonadotropin (eCG), formerly called preg­nant mares’ serum gonadotropin (PMSG) secreted from a specialised endometrial cups. The eCG acts like FSH and LH can induce follicular development and ovulation. The endometrial cup formation occurs around the 35th day of gestation as a girdle-like band of specialised cells developed from the foetal trophoblast and embedded within the uterus after its detachment from the foetal trophoblast. The second­ary CL secretes progesterone from day 80 to nearly mid of pregnancy, day 130-150 of gestation. The placenta is the source of progesterone for the remainder of gestation in these species. Oxytocin and PGF2α induce the regression of CL in mares only when they have functional luteal tissue.

22.3.6 Ovulation in Birds

Female chicken that attains puberty is called a pullet. The sexually matured pullet capable of forming eggs or ova is called an adult or hen. A pullet or hen contains nearly 4000 ova or oocytes in their left ovary, and a few of them are surrounded by different nutrients and gradually become a yolk in their sac or follicle. The follicles are continuously migrated towards the outer circumference of the ovary. The yolk and ovum rupture from the follicles and dissociates from the stalk after acquiring sufficient yolk or nutrients for the chick. This process is called ovulation. Only one ovum can be ovulated at a time. The diameter of a matured pre-ovulating follicle is about 40 mm in fowl. Each follicle requires nearly 10 days time duration in fowl to become mature before ovulation when it reaches about 30-31% weight of the whole egg.

In birds, the follicles of various developmental stages and blood vessels and nerves are suspended together from the ovary. It is called a stalk or pedicle. The domestic fowl generally takes 9 days to develop a fully matured oocyte. The funnel-like infundibulum of the oviduct captures the yolk after ovulation. Then it passes through different parts of the oviduct before laying. The term oviposition refers to the process of laying or shedding completely developed eggs. Various egg layers and shells are formed over the yolk in different parts of the oviduct to become a complete egg (Table 22.25) having species-specific shape and colour (discussed in detail in an earlier chapter). Time taken from ovulation to oviposition depends upon the species (discussed in detail in an earlier chapter). It takes nearly 24-25 h for all

Table 22.25 Role of different parts of the oviduct in egg formation

domestic birds. Thus a bird can’t lay two successive eggs in a day. Two consecutive ovulations can occur in fowl, turkeys and Japanese quail with an interval of 15-30 min and duck and guinea fowl slightly earlier (15 min). The post-ovulatory follicles may remain up to 24 h in fowl and then regress without forming corpus luteum.

22.3.6.1 Control of Ovulation

22.3.6.1.1 Neuro-endocrine Axes

Avian reproductive and ovulation are regulated by the synchronised interactions of different hormones (Table 22.26) and peptides. The central neuroendocrine axis is the hypothalamic-pituitary-gonadal (HPG) axis. The hypo­thalamus integrates the upregulation of the axis and the secretion of GnRH-FSH-LH, followed by ovarian steroids and controls the reproduction. The GnRH neurones are stimulated by environmental factors like photoperiod, seasons, food availability, and specific reproductive signals like courtship, sound or song behaviour. Most birds are seasonal breeders, and the photoperiod influences their

Table 22.26 Role of various hormones in avian female reproduction

breeding. Long photoperiod and favourable seasons upregulate the HPG axis as it facilitates access to food, favouring gaining energy. Stress-induced suppression of reproductive activity is mediated through GnIH after activating the hypothalamic-pituitary-adrenal (HPA) axis. Avian reproduction is also regulated by the hypothalamic- pituitary-thyroid (HPT) axis.

22.3.6.1.2 FSHandLH

The FSH and LH secreted from the anterior pituitary gland under the influence of GnRH play a predominant role in the ovulation process. The FSH regulates follicular growth and maintains the follicular hierarchy. A single ovum is transformed into a pre-ovulatory follicle from 7 to 10 hierar­chical follicles after a rapid growth phase of a small follicular pool. The LH stimulates both the hierarchial and non-hierarchical follicles for steroidogenesis. Androgen and estrogens are produced from the thecal layers of the small follicles and progesterone from the granulosa cells of the pre-ovulatory follicles and small follicles. But, progesterone is converted into androgen and or estrogen when it reaches the theca layer. The production of androgen and estrogen gradually decreased in the pre-ovulatory follicle with the progression of follicular size due to reduced receptivity of FSH receptors in the thecal cells. Thus, the matured pre-ovulatory follicle is capable of only production of pro­gesterone. Progesterone causes a positive feedback loop to stimulate the profuse release of LH from the anterior pitui­tary, resulting in the LH surge and ovulation. The highest inhibin secretion occurs in the large pre-ovulatory follicle and inhibits the next ovulation by down-regulating FSH secre­tion. All the steroids are involved in follicular development and the oviduct development along with up-and down-regu­lation of the HPO axis.

22.3.6.1.3 Prolactin

Prolactin promotes broodiness characteristics as well as nesting behaviour. Its level is increased immediately after oviposition and thus reduces the next ovulation. Prolactin is induced to increase vasoactive intestinal polypeptide (VIP) and reduce gonadotropin-releasing hormone (GnRH) from the pre-optic area (POA) of the hypothalamus. It results in decreased secretion of LH from the anterior pituitary. The level reduces when chicks are self-dependent on food. The secretion of prolactin is controlled genetically. Its level is more in indigenous or non-descript breeds of fowl, and mostly all wild birds show more broodiness characteristics than layer varieties of breeds.

22.3.6.1.4 Thyroid Hormones and Parathyroid Hormone

The nuclear receptors (TRα and TRβ0) and plasma mem­brane receptors (integrin, αVβ3) of both thyroxine (T4) and triiodothyronine (T3) are present in the ovarian follicular cells. The T3 inhibits the ovulation process by inducing atresia of pre-ovulatory follicles, decreasing estrogen levels by reducing the activity of thecal cells of the pre-ovulatory follicles and non-hierarchical follicles, and diminishing the function of LH. But it can increase progesterone levels by influencing the granulose cells of the pre-ovulatory follicle. Thyroid hormones induce the moulting and hatching process; thus, referred to as the hatching hormone. The parathyroid hormones and parathyroid hormone-related proteins promote the relaxation of the proximal oviducts. They also facilitate increased blood flow at the oviduct and surrounding gland to release various egg-forming materials, including calcium.

22.3.6.1.5 IGF

The insulin-like growth factors (IGF-I and IGF-II), synthesis in the ovary, enhance FSH and LH’s receptivity over the thecal and granulosa cells. However, IGF-II augments the recruitment of follicles, and the follicles cannot respond to IGF-II stimulation atretic. Conversely, the urokinase enzyme is active only in small rapid, growing follicles and expressed little in larger follicles. Thus, urokinase is also considered one of the follicular hierarchy determinants.

22.3.6.1.6 Prostaglandins

Prostaglandins involve in the ovulation and egg formation process. It helps to rupture the stigma and facilitates ovula­tion. The PGF2α acts over the shell gland to secrete materials for forming the eggshell. It also helps contract the oviduct to move the ova downward. In reverse, the PGE2 acts over the uterovaginal sphincter to relax the oviduct, resulting in ease of laying.

22.3.6.1.7 Vasotocin

Vastocin belongs to the vasopressin family peptide, involves uterine contraction and regulates oviposition. It also influences social and reproductive behaviours, osmoregula­tion and glycogenolysis.

22.3.6.2 Factors of Ovulation

Genetics (species and breed) (Table 22.27) and nutritional state are the major factors affecting ovulation.

The indigenous or non-descript birds lay less number of eggs in a year. Layer birds are genetically capable of produc­ing more eggs, and various genetic manipulations developed for commercial layer birds. About 2021 and 2623 genes identified in low egg-producing hens (LEPH) and high egg-producing hens (HEPH) affect the HPO axis and regulate the laying process.

Ovulation is delayed when ova takes a long time to receive its optimum nutrients from the surroundings. Thus, poor nutrition causes delayed ovulation. A good hen having good genetic make-up with the optimum level of nutrition

Table 22.27 Occurrence of ovulation in a year of some domestic pure breed birds

Data compiled from various sources

may produce about 300-310 eggs in a year. The size and weight of the ova or eggs are also genetically altered. The size and weight of quail egg is about 3.5 cm (length) ? 2.7 cm (diameter) and 10 g; while 22-11.5 cm (length) ? 18 cm (diameter) having about 140 g weight found in goose egg. A chicken egg’s average size and weight is about 6.2 cm (length) ? 4.3 cm (diameter) and 55 g.

22.3.6.3 Clutch

The birds generally lay eggs in a group, successively for a few days, called a clutch, following an asynchronous gap or pause (break). Clutch is comparable with the litter size of mammals. Length of clutch and pause varies from 1 or 2 to 100 depending on species, breed and age. Habitat, latitude, food availability, and the presence of predators influence the clutch size. The nest supports the brood of the eggs; hence, nesting birds have a comparatively larger clutch size. Wild migratory birds have low clutch size (1-2 eggs) compared to non-passerines like ducks and geese as many as 20 eggs. Birds having a long pre-pubertal period (4-5 years), like marine birds, have a low clutch. Marine birds that scavenge far from their habitat have smaller clutches (one egg) than those which explore near the colony (2-3 eggs). Higher latitudes favour larger clutches as the birds can gather more food per unit of time. Birds of tropical non-seasonal rain­forest and nearer to the equator contain smaller and uniform clutch sizes throughout the year due to a shortage of food compared with the same species and breeds of the polar zone during the spring and summer. Extreme weather and global warming reduce the eggs’ viability, thus reducing the clutch size. More extended day length increases clutch size as extended photoperiod facilitates to search for more food. Daily total exposure to light (including artificial light) requires about 14-16 h in fowl and about 14-18 h in quail to maintain optimum clutch size. Older birds have larger clutch sizes than young ones. Younger birds generally take 1-2 weeks after their first laying to gain a stable clutch size. Peak clutch size often occurs within 3-5 weeks from the day of 1stlaying or attainment of puberty in fowl. Puberty may attain in fowl at about 4-6 months, and they can lay eggs for up to 3 years or more; but, after 1.5 years, the clutch size is reduced, and the break period becomes longer.

22.3.6.4 Formation of Defective Eggs

Various defective eggs may occur due to defects in ovulation and egg formation processes (Table 22.28). Simultaneous ovulation of two ova results in a double-yolk egg. Blood spots within the egg may occur due to the rupturing of blood vessels in the ovary and oviduct. Deficiency of vitamin A and vitamin K in feed, presence of lucerne and fungal toxin in feed and use of specific drugs, like sulphaquinoxaline, may cause such spots. A lack of carotenoids may lead to pale yolk formation. Watery whites or albumin may be occurred due to fungal toxins in the feed, ammonia in the shed and certain diseases like infectious bronchitis and egg drop syndrome. Greenish coloured albumin may form due to the excess use of riboflavin and cyclopropene fatty acids in cottonseed. Any stress develops various grooves over the eggshell, called the misshapen egg. The deficiency of vitamin-D3 and calcium and salt-rich drinking water will cause thin shell eggs and different deformed eggs. Young birds may produce such deformed eggshells due to immature shell glands.

Table 22.28 Various egg deformities with their origin

Source: Das and Roy (2016)

Learning Outcomes

• Endocrinology of female reproduction: The reproductive activity of females is controlled by the hypothalamic-pituitary-ovarian (HPO) axis comprising of hypothalamus, the anterior pituitary, and the ovaries. This HPO axis is responsible for regulating both centrally and peripherally produced reproductive hormones. The central part of the axis includes GnRH from the hypothalamus and gonadotropins from the anterior pituitary, LH and FSH. Ovaries act as dynamic endocrine glands that secret steroid and peptide hormones. The ovaries have two important steroid hormones: estrogens (estradiol, estrone, and estriol) and progesterone (progestin). Activins, inhibins, and follistatin are the peptide hormones produced in the ovaries. All these hormones act in a coordinated fashion to con­trol different reproduction events in animals. The secretion of GnRH is controlled by neuropeptides such as kisspeptin and RFRP3. HPO axis is influenced by several factors like nutrition, photo­period, and stress. Besides these reproductive hormones, pheromones are also involved in regulating sexual behaviour. In mammals, pheromones are released through urine, faeces, vag­inal secretion, saliva and modified scent (cutaneous) glands, including hair and wool. Animals perceive the pheromones through vomeronasal organs and express characteristics of sexual behavioural patterns.

• Puberty and estrous cycle: Puberty is the ability of the animal to produce gamete, i.e. ovum in females. The onset of puberty results from integrated sequences of biological events that lead to progres­sive maturation of sexual characteristics to attain full reproductive capacity. Puberty results due to the activation of the HPO axis. Estradiol suppresses GnRH secretion through a negative feedback mech­anism. At the initiation of puberty, the negative feedback of estradiol is decreased, leading to activa­tion of the GnRH surge centre and commencement of the estrous cycle, growth of the follicles, and ovulation. The rhythmic sexual behavioural pattern exhibited by the female animals after the attainment of puberty is called the estrous cycle. The estrous cycle classifies into four distinct phases: estrus, metestrus, diestrus, and proestrus. During the proes- trus and estrus, follicular development or generation of follicular wave(s) occurs. Hence, these two phases are collectively called the follicular phase. The metestrus and diestrus phases are characterised by the formation of the corpus luteum and are called the luteal phase. The length of estrous cycle, dura­tion of estrus, and time of ovulation are species specific. The estrus cycle is characteristically differ­ent from the menstruation cycle found in primates and humans.

• Oogenesis and folliculogenesis: The developmen­tal process of the female gamete or ovum is called oogenesis. Oogenesis is initiated at the embryonic stage and completed after puberty in three different phases: oocytogenesis, ootidogenesis, and matura­tion (oogenesis proper). The ovarian follicles are the functional units of the ovary that appear as a cluster of somatic cells to protect and nourish the oocytes. The follicular cells are developed through a process termed folliculogenesis. The folliculogenesis pro­cess encompasses the growth and development or atresia of follicles through morphological and func­tional changes. Several hormone and growth factors are involved in the oogenesis and folliculogenesis process.

• Ovulation and corpus luteum formation: Ovula­tion is the biological process that involves the shed­ding of the oocyte from the mature Graafian follicle. It is an inflammatory process sequentially controlled by the neuroendocrine system. Two subsequent events occur at the oocytes and surrounding follicles during the ovulation process. In oocytes, the resumption of meiosis and the structural remodelling of the follicles release the maturing oocyte. The temporary heterogeneous endocrine structure comprises steroidogenic luteal cells, fibroblasts, endothelial, pericytes, and immune cells formed in the ovum-free follicle on the ovarian surface called corpus luteum. It plays a central role in regulating the reproductive cycle and mainte­nance of pregnancy by secreting progesterone. The process of formation of corpora lutea is known as luteinisation. The corpus luteum has two fates depending on the occurrence of fertilisation. If fertilisation and implantation occur, the CL persists throughout the pregnancy as corpus luteum graviditatis. The CL is regressed to form corpus albicans if fertilisation doesn’t happen. The life span of the corpus luteum is governed by the synchronised activity of the pituitary, ovary, uterus, and the embryo through LH, progesterone, oxyto­cin, and prostaglandin. Prostaglandin F2α is the principal luteolytic substance in domestic animals.

• Ovulation in birds: The term oviposition refers to the process of laying or shedding completely developed eggs. The yolk and ovum rupture from the follicles after acquiring sufficient yolk or nutrients for the chick through the ovulation process and captured by the infundibulum. Various egg layers and shells are formed over the yolk in differ­ent parts of the oviduct to develop a complete egg. Several neuroendocrine factors are involved in the ovulation process of birds. The birds generally lay eggs in a group, successively for a few days, called a clutch, following an asynchronous gap or pause (break).

(continued)

Exercises

Objective Questions

Q1. What are the products of meiosis-I?

Q2. Which gonadotropins are mainly responsive to which kind of cells of the oocyte?

Q3. Which kind of antral follicles secrete gonadotropin surge-attenuating factor?

Q4. Why does ovulation occur after mating in induced ovulation?

Q5. Which ovary is more functional in ruminants?

Q6. How does follistatin acts on FSH?

Q7. Which follicles are termed subordinate follicles?

Q8. Which hormone plays the major role in meiotic resumption?

Q9. What is the role of StAR in steroidogenesis?

Q10. How do estrogens perform their anti­inflammatory role?

Q11. How many carbons are in the progesterone skeleton?

Q12. Which physiological phenomenon will occur when the stimulatory mechanism between the kisspeptin and NKB networks with the GnRH surge centre is developed?

Q13. Write the role of resistin in steroidogenesis.

Q14. How many days generally a follicle will take to mature before ovulation in fowl?

Q15. Write the difference between diestrous and diestrus.

Q16. Which reflex cause the occurrence of standing estrus behaviour?

Q17. Why does follicular wave not occur during pregnancy? Q18. Anestrus persisted without occurring estrous cycle is termed as______.

Q19. Write a few major physiological reproductive adaptations in females of small animals.

Q20. Write the name of the specific protein that causes the Bruce effect.

Subjective Questions

Q1. Write the major differences between spermatogenesis and oogenesis.

Q2. Write the factors to control oogenesis and folliculogenesis.

Q3. Write the interrelationship between gonadotropins and ovarian steroid hormones.

Q4. Write the mechanism of ovulation.

Q5. Describe the events in the follicular wave.

Q6. Write the various steps of luteolysis in a cow.

Q7. Why granulosa cells can produce estrogens, but theca cells can’t?

Q8. Write the role of hormones and growth factors in female reproduction.

Q9. Write the various factors affecting puberty.

Q10. Write in brief the role of gonadotropins and ovarian steroids in estrous cycle of an ewe.

Answer to Objective Questions

A1. Secondary oocyte and first polar body

A2. Granulosa cells are responsive to FSH and theca cells to LH

A3. Small antral follicles

A4. LH surge is mating-induced

A5. Right ovary

A6. By reducing the responsiveness of the receptors for activin

A7. The unsuccessful antral follicles become dominant follicles, followed by regression

A8. LH (Surge)

A9. Transport cholesterol to the inner mitochondrial mem­brane from the outside

A10. By mobilising the polymorphonuclear leukocytes or neutrophils

A11. 21

A12. Puberty

A13. Decrease steroidogenesis

A14. 10 days

A15. Diestrous means occurring of two estrous cycles in a year, and diestrus is a phase of estrous cycle

A16. Lordosis reflex

A17. Due to the lack of FSH

A18. Primarypersistentanestrus

A19. Earlier puberty, more litter size, short gestation, very less duration of lactation anestrus

A20. Major histocompatibility complex (MHC) class 1 protein

Keywords for the Answer to Subjective Questions

A1. Oogonium and spermatogonium, duration, stage of gamete production

A2. Gonadotropins, sex steroid hormones, several proteins

A3. Relationship between FSH and LH with estrogens and progesterone

A4. LH surge, role of proteolytic substances, production of local paracrine effectors

A5. Changes of follicles up to dominant follicle from the ovarian pool, atresia, hormonal changes

A6. Role of PGF2α, oxytocin, various tissue degenerating substances, and cytokines

A7. Two cells two gonadotropins, presence of specific enzymes in both the cells

A8. Role of primary, secondary, and tertiary hormones

A9. Breed, age and body weight, nutrition, endocrines, and growth factors and environment

A10. Role of FSH, LH, estrogen, and progesterone in estrus, metestrus, diestrus, and proestrus

Further Reading

Textbook

Conti M, Chang RJ (2016) Endocrinology: adult and pediatric, vol II, 7th edn. Elsevier, Philadelphia, PA, pp 1-77. ISBN: 978-0-323­18907-1.

Das PK, Roy B (2016) Eggopedia. Parul Prakashani Pvt Ltd., pp 1-150. ISBN: 9789385555961.

Gougeon A (2004) Chapter 2: Dynamics of human follicular growth: morphologic, dynamic, and functional aspects. In: The ovary, 2nd edn. Academic Press, pp 25-43. https://doi.org/10.1016/B978- 012444562-8/50003-3

Mucignat-Caretta C (2014) Chapter 16: Cattle pheromones, Table 16-1: Cattle pheromones: identification, source, and functions. In: Neuro­biology of chemical communication. CRC Press/Taylor & Francis, Boca Raton, FL

Research Articles

Oogenosis and Folliculogensis

Alwan AF, Al-Saffar HE, Khammas DJ (2005) Biometry of genital organs in Iraqi female buffalo. Iraqi J Vet Sci 19(1):77-81. https:// doi.org/10.33899/ijvs.2005.37421

Bachler M, Menshykau D, ChDe G et al (2014) Species-specific differences in follicular antral sizes result from diffusion-based limitations on the thickness of the granulosa cell layer. Mol Hum Reprod 20(3):208-221. https://doi.org/10.1093/molehr/gat078

Brady K, Liu HC, Hicks JA et al (2020) Transcriptome analysis of the hypothalamus and pituitary of turkey hens with low and high egg production. BMC Genomics 21(1):647. https://doi.org/10.1186/ s12864-020-07075-y

Griffin J, Emery BR, Huang I et al (2006) Comparative analysis of follicle morphology and oocyte diameter in four mammalian species (mouse, hamster, pig, and human). J Exp Clin Assist Reprod 3(2). https://doi.org/10.1186/1743-1050-3-2

Haque Z, Haque A, Quasem MA (2016) Morphologic and morphomet­ric analysis of the ovary of black Bengal goat (Capra hircus). Int J Morphol 34(1):13-16

Hoque MSA, Islam MM, Selim MAS (2016) Interspecies Differences on ovarian parameters between black Bengal goat and indigenous Bengal sheep in view of In vitro maturation. Adv Life Sci 6(3):54-60. https://doi.org/10.5923Zj.als.20160603.02

Monniaux D, Clement F, Dalbies-Tran R et al (2014) Theovarian reserve of primordial follicles and the dynamic reserve of antral growing follicles: what is the link? Biol Reprod 90(4):85, 1-11. https://doi.org/10.1095/biolreprod.113.117077

Ptak G, Matsukawa K, Palmieri C et al (2006) Developmental and functional evidence of nuclear immaturity in prepubertal oocytes. Hum Reprod 21(9):2228-2237. https://doi.org/10.1093/humrep/ del184

Reynaud K, Gicquel C, Thoumire S et al (2009) Folliculogenesis and morphometry of oocyte and follicle growth in the feline ovary. Reprod Domest Anim 44(2):174-179. https://doi.org/10.1111/j. 1439-0531.2007.01012.x

Sasan JS, Uppal V, Bansal N et al (2016) Histological exploration of Graafian and atretic follicles of buffalo ovary: a seasonal study. Buffalo Bull 35(1):135-146

Songsasen N, Fickes A, Pukazhenthi BS et al (2009) Follicular mor­phology, oocyte diameter and localisation of fibroblast growth factors in the domestic dog ovary. Reprod Domest Anim 44(Suppl 2):65-70. https://doi.org/10.1111/j.1439-0531.2009.01424.x

Corpus Luteum, Puberty—Maturity, Steriodogenesis and Estrous Cycle

Calejja-Agius J, Muttukrishna S, Jauniaux E (2009) Role of TNF-α in human female reproduction. Expert Rev Endocrinol Metab 4(3):273-282. https://doi.org/10.1586/eem.09.4

Casarini L, Crepieux P (2019) Molecular mechanisms of action of FSH. FrontEndocrinol 10:305. https://doi.org/10.3389/fendo.2019.00305

Dobrzyn K, Smolinska N, Kiezun M et al (2018) Adiponectin: a new regulator of female reproductive system. Int J Endocrinol 2018: 7965071. https://doi.org/10.1155/2018/7965071

Hansen PJ (2019) Reproductive physiology of the heat-stressed dairy cow: implications for fertility and assisted reproduction. Anim Reprod 16(3):497-507. Epub 28 Nov 2019. https://doi.org/ 10.21451/1984-3143-ar2019-0053

Hu KL, Chang HM, Li R et al (2019) Regulation of LH secretion by RFRP-3—from the hypothalamus to the pituitary. Front Endocrinol 52:12-21. https://doi.org/10.1016/j.yfrne.2018.03.005

Jones ASK, Shikanov A (2019) Follicle development as an orchestrated signaling network in a 3D organoid. J Biol Eng 13:2. https://doi.org/ 10.1186/s13036-018-0134-3

Khan A, Khan MZ, Umer S et al (2020) Cellular and molecular adapta­tion of bovine granulosa cells and oocytes under heat stress. Animals (Basel) 10(1):110. https://doi.org/10.3390/ani10010110

Kowalewski MP (2012) Endocrine and molecular control of luteal and placental function in dogs: a review. Reprod Domest Anim 47 (Suppl. 6):19-24. https://doi.org/10.1111/rda.12036

Kowalewski MP (2014) Luteal regression vs. prepartum luteolysis: regulatory mechanisms governing canine corpus luteum function. Reprod Biol 14:89-102. https://doi.org/10.1016Zj.repbio.2013. 11.004

Lucy MC (2019) Stress, strain, and pregnancy outcome in postpartum cows. Anim Reprod 16(3):455-464. https://doi.org/10.21451/1984- 3143-ar2019-0063

Prins JR, Gomez-Lopez N, Robertson SA (2012) Interleukin-6 in preg­nancy and gestational disorders. J Reprod Immunol 95(1-2):1-14. https://doi.org/10.1016/j.jri.2012.05.004

Schuermann Y, Rovani MT, Gasperin B, Ferreira R, Ferst J, Madogwe E, Gongalves PB, Bordignon V, Duggavathi R (2018) ERK1/2-dependent gene expression in the bovine ovulating follicle. Sci Rep 8:16170. https://doi.org/10.1038/s41598-018-34015-4

Tsatsanis C, Dermitzaki E, Avgoustinaki P et al (2015) The impact of adipose tissue-derived factors on the hypothalamic-pituitary-gonadal (HPG) axis. Hormones 14(4):549-562. https://doi.org/10.14310/ horm.2002.1649

Wani A, Dhindsa SS, Tawheed AS, Chowdhary SRA, Balwinder K (2013) The role of pheromones in animal reproduction - a review. Progress Res 8(1):14-18

Zubeldia-Brenner L, Roselli CE, Recabarren SE et al (2016) Develop­mental and functional effects of steroid hormones on the neuroendo­crine axis and spinal cord. J Neuroendocrinol 28(7):10. https://doi. org/10.1111/jne.12401

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