Endocrinology of Female Reproduction

22.1.1 Hypothalamic-Pituitary-Ovarian Axis

The reproductive activity of females is regulated by the hypothalamic-pituitary-ovarian (HPO) axis comprising the hypothalamus, the anterior pituitary, and the ovaries (Fig.

Fig. 22.1 Hypothalamus­Pituitary-Ovary (HPO) axis. Figure shows the interrelationship among the hypothalamus, pituitary gland, and ovary. The hypothalamus releases GnRH (gonadotropin-releasing hormone), which acts on the pituitary gland to secrete gonadotropins, FSH (follicle­stimulating hormone), and LH (luteinising hormone). The FSH acts on the growing follicles and stimulates their growth to become antral follicles. LH acts on the mature follicle and causes rupturing of the follicle or ovulation, followed by the formation of the corpus luteum. The first two steps, the follicular development and development of mature follicle, are considered the follicular phase, where released E2 (estrogen) and activin positively stimulate the axis to release gonadotropins (illustrated by the solid positive (+) lines). The release of FSH from the pituitary gland is suppressed in the follicular phase (shown by the dotted negative (-) lines) by the inhibin, follistatin. The FSH secretion is also negatively controlled by excess secretion of E2 in the follicular phase, and secretion of P4 (progesterone) in the luteal phase, comprising ovulation and corpus luteum formation

hormones. The central part of the axis includes GnRH, secreted from the hypothalamus, and gonadotropins, viz. LH and FSH are released from the anterior pituitary.

22.1.1.1 Hypothalamic GnRH Secretion

GnRH is a decapeptide produced in the hypothalamus to regulate the release of two gonadotropins, namely LH and FSH, from the anterior pituitary.

The secretion of GnRH is controlled by some small peptides called RFamides. They are so named due to the Arg-Phe-NH2 motif at the C-terminus. Two groups of RFamide peptides, namely kisspeptins and Gonadotropin­Inhibiting Hormone (GnIH), have stimulatory and inhibitory roles in GnRH secretion, respectively. The GnRH-secreting neurones are co-localised with the kisspeptin-neurokinin B- dynorphin (KNDy) neuronal network. The kisspeptin- secreting neurones in the hypothalamus are identified in the anteroventral periventricular nucleus (AVPV), the rostral periventricular region of the third ventricle (RP3V), periventricular nucleus (AVPV), and arcuate nucleus in case of rodents. The predominant site for kisspeptin-secreting neurones in ruminants is the arcuate nucleus. In humans, the kisspeptin neurones are localised at the rostral pre-optic area (POA) and in the infundibular nucleus. Kisspeptin is a potent stimulator of the HPO axis. It stimulates GnRH-secreting neurones by activating protein kinase C involving inositol triphosphate and diacylglycerol pathway. The RFamides were found to decrease gonadotropin secretion in a dose­dependent manner, so designated as GnIH.

22.1.1.2 Pituitary Gonadotropin Secretion

The anterior pituitary secretes two gonadotropins from its gonadotroph, FSH and LH, glycoproteins in nature. The frequencies of GnRH pulses determine the secretion of FSH and LH. Slow GnRH pulse frequency (< 1 pulse per 2-3 h) increases FSH secretion by augmenting FSH-β gene tran­scription. In contrast, a rapid GnRH pulse (>1 pulse per h) increases LH-α and LH-β gene transcription causing the release of LH. The LH pulse frequency increases during the follicular phase, particularly in the pre-ovulatory period and gradually declines during the luteal phase. The FSH stimulates follicular growth and estradiol formation by induc­ing the aromatase enzyme. At the late stages of the follicular phase, activins and estradiol enhance the actions of FSH. LH facilitates ovarian steroidogenesis in the pre-ovulatory follicles. The theca and granulosa cells of the ovary are stimulated independently by LH and FSH. The ovarian ste­roidogenesis in the pre-ovulatory follicle is mediated through LH receptors on theca and FSH receptors on granulosa cells (see ‘two cell two gonadotropin theories’). The steroidogenic acute regulatory protein (StAR protein) facilitates andro­stenedione production, which serves as an estrogen precursor after its diffusion to the granulosa cells.

22.1.1.3 Ovarian Hormones

Besides producing mature oocytes, ovaries act as dynamic endocrine glands that secret steroid and peptide hormones and play pivotal roles in female reproduction. 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.

22.1.1.3.1 Steroidogenesis

Cholesterol is the prime precursor of all steroid hormones. Steroidogenesis in the ovaries requires the delivery and uptake of cholesterol precursors and their conversion into estrogen and progesterone through a series of enzymatic reactions catalysed by steroid cytochrome P450 (CYP) hydroxylases and hydroxysteroid dehydrogenases.

22.1.1.3.1.1 Cholesterol Uptake

Cholesterol can’t dissolve in the body fluid due to its hydro­phobic nature and is carried through lipoproteins. In cattle, pigs, and humans, cholesterol is incorporated by the steroido­genic cells in low-density lipoprotein (LDL). The follicular and luteal cells of the ovaries have lipoprotein receptors, namely scavenger receptor class B member 1 (SR-BI). It binds with LDL and incorporates cholesterol inside the cells as lipid droplets in the form of cholesterol esters. The choles­terol ester hydrolase enzyme converts the cholesterol esters to free cholesterol in the cytoplasm of these cells. This free cholesterol carries from the outer to the inner mitochondrial membrane through steroidogenic acute regulatory protein (StAR). This step is the rate-limiting step of steroidogenesis.

22.1.1.3.1.2 Steroidogenesis

In the mitochondria of the ovarian cells, free cholesterol is converted to pregnenolone by the enzyme cytochrome P450 (a cholesterol side-chain cleavage enzyme, P450scc, CYP11A1). The pregnenolone then diffuses into the smooth endoplasmic reticulum, where it converts to progesterone by the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD).

22.1.1.3.1.3 Steroidogenesis in Corpus Luteum

The luteal cells have the receptors for LH, responsible for upregulation of StAR and LDL in the luteal cells along with stimulation of P450scc.

22.1.1.3.1.4 Steroidogenesis in the Placenta

In sheep, horses, cats, guinea pigs and humans, the placenta, apart from the ovaries, plays a pivotal role in maintaining the pregnancy by producing progesterone. The trophoblast cells are mainly responsible for steroidogenesis (androgens and

Fig. 22.2 The chemical structures of estrogens. Estrone (E1) has single hydroxyl (-OH), Estradiol (E2) has two hydroxyls, Estriol (E3) has three hydroxyls, and Estetrol (E4) has four hydroxyls. Estrone is synthesised from androstenedione by the enzyme aromatase. It is also produced reversibly from estradiol by the enzyme 17β-hydroxysteroid dehydrogenase (17β-HSD) in various tissues like the ovary, liver, uterus, and mammary gland. Estradiol is chiefly available in females

estrogens) in cows, sheep, pigs, and rats. In these species, the enzyme 17α-hydroxylase (Cytochrome P450 17A1,

CYP17A1) mainly controls the steroidogenesis process. In species such as horses, rhesus monkeys, and humans, the placenta is devoid of these enzymes; hence, the gonadal interstitial cells and adrenal gland provide the androgen for the production of placental estrogens in horses and primates, respectively. The placenta of the horse and monkey secretes both estrogens and progesterone, but the placenta of mouse and rabbit secretes only estrogen. In the rat, ovarian estrogens suppress the activity of 17α-hydroxylase in the placenta, but placental androgens act as the precursors of ovarian estrogens for aromatisation.

22.1.1.3.2 Estrogens

22.1.1.3.2.1 ChemicalStructure

All estrogens are chemically made of estrane skeleton (C-18), known as C-18 steroids (Fig. 22.2). The aromatisation of the estrogens’ A-ring leads to a planar structure where all carbons are in the same plane.

produced from androstenedione/testosterone by aromatisation in the granulosa cells of the ovarian follicle; beyond that, it can be produced in adrenal glands, fat, liver, the breasts, and brain. Estriol is mostly found in pregnant females produced in the placenta. Estetrol is an intermediate product, synthesised during pregnancy only in the foetal liver from estradiol (E2) and estriol (E3) by the actions of two enzymes, 15α-hydroxylase, and 16α-hydroxylase

22.1.1.3.2.2 Source

Estrogen is mainly synthesised in the granulosa cells of the ovary (Fig. 22.3). The extragonadal sources of estrogens are the placenta, adrenal glands, and brain. Adipocytes also secret a small amount of estrogens that originate from the conversion of peripheral androstenedione to estrone. Phytoestrogens are the estrogenic compounds available in various plants and feed sources such as alfalfa, red clover, white clover, subterranean clover, berseem clove, and the seed of soybean, sunflower, sesame, etc. The estrogens are subjected to gastrointestinal and hepatic inactivation; hence, difficult to apply in the oral route. Estrogens are also synthesised in bulls, boars, stallions, dogs, and men testes. Equilin and equilenin are the two estrogenic compounds produced in the mare placenta.

22.1.1.3.2.3 Transportation and Activation

Estradiol, like other steroids, is hydrophobic and circulates in conjugation with plasma proteins. Only 1-2% estradiol is circulated in free form. Estradiol has a high affinity to binding with sex hormone-binding globulin (SHBG) synthesised in the liver. But, due to less availability of globulin, nearly 40% of estradiol is transported in conjugation with SHBG and the remaining portion is circulated after binding with plasma albumin. However, albumin has less affinity for estradiol.

22.1.1.3.2.4 MechanismofAction

Estrogens act through estrogen receptors, namely ER-α and ER-β. Both these receptors are present in the ovaries. But ER-β is predominant in the granulosa cells, and ERa-α is

Fig. 22.3 Two cell two gonadotropins. Figure shows steroidogenesis in the ovary. Two gonadotropins, the FSH (follicle-stimulating hor­mone) and LH (luteinising hormone), are released from the pituitary gland. The receptor for FSH presents in granulosa cells, and receptors for the LH are present in both granulosa and theca cells. The FSH and LH stimulate adenylate cyclase and produce cAMP (cyclic adenosine monophosphate) to activate protein kinase A utilising adenosine tri­phosphate (ATP), GTP (guanosine triphosphate) and GDP (guanosine diphosphate). The protein kinase A, in turn, stimulates the various enzymes required for steroidogenesis (shaded arrow). The cholesterol is utilised by the CYP11A1 (cytochrome P450, a cholesterol side-chain cleavage, P450scc) to produce pregnenolone that can be diffuse to both thecal and granulosa cells (reversible arrow) and converted to proges­terone or DHEA (dehydroepiandrosterone) as well as androstenedione by the enzymes 3β-HSD (3β-hydroxysteroid dehydrogenase) and CYP17 (cytochrome P450). The androstenedione is further converted into testosterone by 17β-HSD (17β-hydroxysteroid dehydrogenase). The end products of cholesterol in the theca cell are androstenedione, and testosterone can transfer to the granulosa cell through the basement membrane, where the influence of aromatase or CYP19A1 produces estradiol and estrone. Thus, the availability of estrogens in the blood is regulated in granulosa cells with the influence of FSH, and its precursors are regulated in the theca cell by the LH

situated in the thecal and luteal cells. There are three pro­posed pathways of estradiol signalling. In the ‘classical path­way’, estradiol binds with its cytosolic receptors (ER-α and ER-β), and the conformational changes (receptor dimerisation) occur. The hormone-receptor complex translocates to the nucleus and binds with estrogen­responsive elements (EREs) in the regulatory regions of estrogen-responsive genes. In a ‘tethered signalling path­way’, estrogen receptors interact with non-ERE response elements to regulate gene expression after binding with tran­scription factors (activating protein-1, AP-1 or the stimulating protein-1, Sp1) other than estrogen. In the ‘nongenomic pathway’, estrogens interact with plasma membrane-bound receptors, initiate cytoplasmic signalling pathways, and activate MAPKs.

22.1.1.3.2.5 Functions of Estrogens

Estrogens have various roles in female reproduction.

Ovarian effects: Estradiol facilitates granulosa cell prolifera­tion by upregulating the expressions of several genes required for granulosa cell differentiation together with FSH and LH in an autocrine or paracrine fashion. Estra­diol also enhances FSH-induced aromatase expression. Estrogens trigger an increased blood flow to the ovaries, thus favours ovulation.

Effects on other reproductive organs: Estradiol regulates the contractility and secretory activity of the cervix, uterus, and fallopian tube by epithelialisation, Vascularisation, and fat deposition together with the oxytocin and prostaglandins. Estrogens enhance the rate of protein syn­thesis and uptake of glucose and water to support the growth of the lining epithelium and underlying muscular tissue (endometrium) of the uterus.

Expression of behavioural estrus and sex desire: It is respon­sible for sexual receptivity or sex desire, i.e. onset of heat (discussed in the estrous cycle).

Roles in gamete transport: Estradiol promotes gamete trans­port (both sperm and ovum) by increasing the contraction of oviductal smooth muscle and ciliary beat frequency (CBF) of the oviduct through the phosphorylation of pro­tein kinase C and A (PKC and PKA) and production of cAMP. Estradiol stimulates the release of antioxidants in the oviductal fluid and reduces sperm stress during sperm transport.

Role in implantation: Estradiol favours the embryo transport from the fertilisation site to the implantation site after stimulating smooth muscle contraction, inducing fluid production and flow, and increasing the CBF.

Immunoprotection of the embryo: Estradiol protects the embryo from the maternal immune system via the activa­tion of ER-α in the oviductal epithelial cells.

Role in parturition: Estrogens cause rhythmic contractions of the uterus during parturition. The E2 and estrone sulphate are mainly available in early pregnancy and late gestation. A higher level of E2, around 24 h before the parturition, helps remove the progesterone block and initiates the parturition (discussed details in parturition).

Development of secondary sexual characteristics: Estrogens are responsible for expressing secondary sexual characteristics in females, viz. hair growth, skin texture, body configuration, voice, etc.

Effects on mammary gland: Estrogen stimulates ductal growth of the mammary gland through ER-α expressed in the mammary epithelium and stroma.

22.1.1.3.2.6 Non-reproductive Roles of Estradiol

Other than reproductive effects, estradiol is also involved in the functioning of different systems like the heart, skin, muscle, bone, brain, and liver.

Estrogens help modulate total cholesterol levels by decreasing LDL or increasing HDL cholesterol through nuclear and extranuclear ER-α and ER-βb by altering the HMG-CoA reductase gene promoter as it contains an estrogen-responsive estrogen element-like sequence (Red-ERE). Decreased level of estrogens leads to atherosclerosis and the risk of a heart attack in postmeno­pausal women.

Estrogens accelerate bones’ linear growth and epiphyseal closure and increase bone density and strength. Hence, skel­etal growth is arrested after puberty and osteoporosis after menopause in females.

Estrogen affects the structure and function of muscle, tendon, and ligaments. Muscle weakness is evident in post­menopausal women due to lower estrogen levels.

Estrogen receptors are present in several brain regions like the hippocampus, cerebral cortex, claustrum, hypothalamus, subthalamic nucleus, amygdala, and thalamus. Estrogens promote cerebral blood flow, neuronal activity, and anti­inflammatory effects in the CNS, thus acting as neuroprotective and neurotrophic agents. The estrogens con­trol sex-specific brain activity. Estrogens also increase sero­tonin levels to influence sexual desire in males and females. In the male, the testosterone in the brain is converted to estrogen by aromatisation. Estrogens influence the sensory inputs of vision, audition, and olfaction and their integration with the motor neurons for muscles of the genital tract to exhibit lordship, mounting, and other sexual behaviour.

Estrogens play a pivotal role in metabolisms such as food intake, glucose homeostasis, lipolysis/lipogenesis, and osmo­regulation. Most of these functions exhibit conjugation with other peptides involved in energy expenditure like leptin, neuropeptide Y, pro-opiomelanocortin, or melanin­concentrating hormone). Estrogens regulate water and salt balance stimulating reabsorption by inducing arginine vaso­pressin (AVP), atrial natriuretic peptide (ANP), renin, and aldosterone. Estrogens stimulate lipid and lipoprotein metab­olism in the liver and upregulate some serum proteins like thrombin and fibrinogen. Therefore, estrogen therapy may lead to venous thromboembolism.

Estradiol indirectly regulates the expression of cardiac heat shock proteins 70 and 72 (HSP70 and 72) and reduces apoptosis by stabilising the mitochondrial membrane and averting apoptosome formation.

Estrogens have anti-inflammatory properties. Estrogen regulates the activity of immune cells through the regulation of cellular metabolism. ERR-α controls the metabolic activity of T cells and influences T cell activation.

22.1.1.3.3 Progesterone

Progesterone is essentially required to establish and maintain pregnancy (pro-gestational) and the reproductive cycle.

22.1.1.3.3.1 Chemical Structure

Progesterone belongs to the class ‘progestogen’, and the molecule has a 21-carbon skeleton called the pregnane skeleton (C-21) (Fig. 19.21). Both natural and synthetic forms of progesterone are available, and synthetic progestogens are usually referred to as progestins.

22.1.1.3.3.2 Source

Major source of progesterone (P4) is the corpus luteum of the ovary in all mammals. The luteal cells contain the enzymes required to synthesise progesterone from the cholesterol (Fig. 22.3). It can also produce in adrenal glands and placenta during pregnancy, particularly in mare, ewe, queen, and human. But the placental progesterone is not sufficient to maintain the pregnancy in cattle, goats, pigs, dogs, and rats. In the case of sheep, horses, cats, and humans, the placental progesterone from the mid-pregnancy is sufficient to support the pregnancy independent of CL. In camels, a large quantity of placental progesterone produces from the multinucleate giant cells by day 30-35 of gestation. The placenta of mare and ewe produces 5α-pregnane instead of progesterone. In cows, progesterone produces from the placenta during pregnancy’s latter half (6-8 months). Placental progesterone is absent in doe, sow, bitch, camel, and rabbit. The growing follicles can produce a small quantity of progesterone in bitch.

The plasma progesterone concentration is highest in the sow, lowest in the ewe and intermediate in the cow. Progester­one secretion sustains throughout the luteal phase of cyclic females. The LH primarily controls progesterone synthesis, and the PGF2α generally destroys the progesterone-producing cells. Hence, progesterone activity is governed by the pulsatile release of pituitary LH and the PGF2α of the uterine endome­trium. Phyto-progesterone is available in the Juglans regia. The Dioscorea mexicana contains progesterone-like steroids (diosgenin), which can act as the precursor of progesterone. Progesterone can also produce in the nervous system. Proges­terone may also be available as the chief transitional substance for circulating androgens and estrogens.

22.1.1.3.3.3 Transportation

About 98-99% of progesterone is transported in protein­bound form. Nearly 80% of progesterone is combined with albumin, 18% with corticosteroid-binding globulin (CBG), or transcortin and less than 1% with SHBG.

22.1.1.3.3.4 MechanismofAction

The progesterone receptor (PR) has two major isoforms, PR-A and PR-B. After ligand binding, the hormone-receptor complex translocates to the nucleus and binds hormone­responsive elements (HRE) at regulatory regions hormone­responsive genes to initiate new protein synthesis. The receptors for androgens, mineralocorticoids, and glucocorticoids can also recognise the HRE of progesterone. Recently, it has been identified that the actions of progester­one are mediated by membrane-localised progestin receptors other than classical PRs (PR-A and PR-B). The membrane­bound progesterone receptors are called the progesterone membrane component (PGRMC1 and 2) and membrane pro­gestin receptors (mPs). The actions of progesterone occur through these receptors and are mediated after the initiation of intracellular signalling pathways and subsequent cellular responses. They can also modulate the genomic actions of progesterone.

22.1.1.3.3.5 Function of Progesterone

Regulation of uterine functions/maintenance of pregnancy: Progesterone plays a central role in maintaining pregnancy by modulating uterine physiology, such as endometrial maturation, reduction of uterine contractility, and favours uteroplacental circulation. It also modulates maternal immune response by suppressing the inflammatory mediators and preventing the foetal allograft’s rejection for supporting pregnancy. Thus, susceptibility to metritis may occur during the postpartum period when progester­one level is diminished suddenly. Progesterone helps dif­ferentiate endometrial stromal fibroblasts into specialised secretory decidual cells that secret uterine milk to provide nutritional support to the embryo before implantation. This process is called decidualisation of the uterus and potentiates placental development.

Regulation of ovarian functions: Progesterone plays pivotal roles in oocyte meiosis, ovulation, luteinisation, and main­tenance of CL. Progesterone helps in meiosis resumption by disrupting gap junctions between cumulus cells. Pro­gesterone inhibits FSH release, thus suppressing follicular growth, ovulation, and estrogen level, which favours the gestation.

Effect on the oviduct: Progesterone stimulates the morphol­ogy and function of the luminal epithelium of the fallopian tube to regulate the volume and composition of the ovi- ductal fluid. It also regulates the muscular activity of the oviduct.

Role in lactation: During pregnancy, progesterone in combi­nation with prolactin favours epithelial proliferation lead­ing to the formation of alveoli. But progesterone inhibits lactation during pregnancy. Copious milk secretion occurs immediately after parturition only when progesterone levels markedly decrease.

Inhibition of sexual behaviour: Progesterone inhibits sexual behaviour. It acts in the ventromedial hypothalamus or the brain’s pre-optic area to block the LH surge and estrogen- induced sexual behaviour. Progesterone inhibits the ion channels and vomeronasal sensory neurons and suppresses pheromone-induced sexual behaviour.

Non-reproductive roles: Progesterone metabolites like di-hydro progesterone (DHP) and 3α, 5α-tetra hydro pro­gesterone (allopregnanolone) are available in the central and peripheral nervous system. They can modulate neuro­nal and astroglial plasticity, enhance neural survivability, support the myelination process, increase neurogenesis in adulthood, and inhibit lipid peroxidation and anti­inflammatory properties. Thus, progesterone therapy is effective in brain injury, ischaemia, and peripheral neuropathy.

Progesterone is reported to increase appetite to facilitate a positive energy balance during pregnancy. A high proges­terone level reduces the natriuresis (lack of sodium retaining aldosterone activity). Thus, extracellular volume is reduced.

Progesterone as contraceptive and application in assisted reproductive technologies (ART): Progesterone is used as a contraceptive as it suppresses follicular development and ovulation by inhibiting FSH secretion. Exogenous proges­terone can be administered through implants (Intrauterine device, IUD, or intravaginal) or oral and parenteral routes. But the most preferred route is vaginal implants. It increases the bioavailability of progesterone by 40-fold more than oral progesterone. The follicular development is suppressed till the withdrawal of progesterone. Upon withdrawal, it results in immediate secretion of FSH followed by follicular development and ovulation within 2-3 days. This functional activity of progesterone is extensively applied to synchronise the ovulation in integrated artificial insemination programme, superovula­tion, embryo transfer technology (ETT), and assisted reproductive technology (ART).

Metabolism and Excretion of Sex Steroids

The half-life of ovarian steroids is less (Table 22.1). Estrogens are metabolised into estrogenically inactive metabolites like estrone and estriol by cytochrome P450 (CYP) enzymes, mainly in the liver. Cytochrome P450 oxi­dase enzymes (CYP3A4 and CYP1A1) cause the oxidation of the 17β-hydroxyl group. The inactive metabolites undergo sulphate and glucuronide conjugation in the liver. Estradiol is excreted via the urine (nearly 75%) or faeces (25%). Estriol is the main estrogen metabolite found in the urine. Progesterone lacks a hydroxyl group; hence, it can’t be easily sulphated and esterified. Thus both the solubility and half-life of

Table 22.1 Biological half-life of steroid hormones

progesterone are more than estrogen. Nearly 60-65% of progesterone is metabolised by 5α-reductase and 5- β-reductase to form allopregnanolone and pregnanolone, respectively. A small amount of progesterone is also metabolised into 11-deoxycorticosterone by 21-hydroxylase. Most progesterone is metabolised in the liver, GI tract (particularly in the duodenum), and kidney. Inactivated progesterones are excreted by the kidney in con­jugated form (through glucuronidation or sulphation).

The urinary concentrations of the inactivated steroid com­pound provide an essential clinical index of reproductive function and determinate the stage of ovarian dynamics. In wild animals, estimation of the inactivated form of steroids from urine and faeces are estimated to evaluate the reproduc­tive status. Some inactive metabolites of estrogens are estrone sulphate, estradiol glucuronide, 2-hydroxy estrone, 2-hydroxy estradiol, 16α-hydroxy estrone, and 16α-hydroxy estradiol. Estrone is also considered a less active form of estradiol. Inactivated form of progesterone is pregnenolone sulphate.

22.1.1.3.4 Control of Ovarian Steroidogenesis

22.1.1.3.4.1 Endocrine Control (Two Cell Two

Gonadotropin Theory)

Ovarian steroidogenesis is controlled by two gonadotropins (FSH and LH) acting on the thecal/luteal cells and granulosa cells, respectively. Hence, it is called the ‘two cell, two gonadotropin theory’. Figure 22.3 depicts the mechanism of the ‘two cell, two gonadotropin theory’. The receptors for FSH are present in the granulosa cell, and receptors for LH are abundant in both granulosa and theca cells. The synthesis of estradiol requires a synergistic relationship between theca cells which produce androgens (i.e. dehydroepiandrosterone (DHEA), androstenediol, androstenedione, testosterone under the influence of LH, and these androgens then diffuse into granulosa cells and converted to estrogens (i.e. estrone, estradiol) by the action of cytochrome P450 aromatase (CYP19A1) stimulated by FSH. LH acts through G protein- coupled receptors after stimulating adenylate cyclase to pro­duce cAMP, which stimulates protein kinase A (PKA). The PKA then vigorously phosphorylates the StAR, transports cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane, and initiates steroidogenesis of androgens (DHAE).

On the other hand, FSH stimulates adenylate cyclase via G protein-coupled receptors. The cyclic adenosine monophosphate (cAMP) generated from adenosine triphos­phate (ATP) activates protein kinase A to stimulate the expression of the respective steroidogenic enzymes such as NADPH cytochrome P450 reductase, which transfers electrons to aromatase; HSD3B2 which converts DHEA to androstenedione, and type 1 17β-hydroxysteroid dehydroge­nase (HSD17B1), the ‘estrogenic’ 17β-HSD that reduces estrone to estradiol. The estrone is produced by the action of cytochrome P450 aromatase (P450arom or aromatase) enzyme and the estrone and subsequently converted to estra­diol by 17β-HSD (CYP19A1) (Fig. 22.3). Hence, granulosa cells are an abundant source of estrogens. Progesterone pro­duction is limited in the granulosa cells due to the lack of the 3β-HSD enzyme. But, in theca cells, pregnenolone can be converted to 17α-hydroxypregnenolone by P45017-OH under the influence of LH. Thus, in the ovary, progesterone can be synthesised with the influence of LH only, but the involvement of both LH and FSH are required for estrogen production. The ovarian theca cells can be compared with the testicular Leydig cells and granulosa cells with the Sertoli cells, considering their steroidogenesis activity.

Activins and inhibins have paracrine effects on ovarian steroidogenesis. The granulosa cell-derived inhibin is stored in antral fluid, diffuses to the adjacent thecal cell layer, and positively stimulates androgen synthesis in LH-stimulated theca cells. Activin stimulates estradiol synthesis by upregulating the aromatase enzyme and FSH receptors.

22.1.1.3.4.2 Molecular Control

Several LH-induced signalling molecules regulate the activ­ity of StAR and control the steroidogenesis (Table 22.2), viz. cAMP and PKA, insulin-like growth factors (IGFs), etc.

22.1.1.3.5 Ovarian Peptide Hormones

22.1.1.3.5.1 Activin

Activins are transforming the growth factor-beta (TGF-β) superfamily of cytokines secreted from the granulosa cells of the ovary. Structurally activins are dimers of inhibin β subunits that act through the classical TGF-β signalling path­way. There are several forms of activins based on the types of

Table 22.2 LH-induced signalling molecules to regulate steroidogenesis

β subunits. They are activin A (β_Aβ_A), activin B (β_Bβ_B), activin C (β_Cβ_C), activin D (β_Dβ_D), and activin E (β_Eβ_E). Activin controls ovarian and testicular development. It stimulates FSH secretion, promoting oocyte maturation and granulosa cell steroidogenesis. Activin has a negative para­crine effect over LH-induced theca cells for androgen pro­duction. It also helps in folliculogenesis by preventing luteinisation of the premature antral follicle. It is also involved in endometrial repair, decidualisation, and preg­nancy maintenance. Other functional features of activins include morphogenesis of the embryo, particularly the devel­opment of the limb’s nervous system and the development of facial and dental structures. In the testis, activins modulate germ cell development and Sertoli cell proliferation.

22.1.1.3.5.2 Inhibin

Inhibins are the glycoproteins composed of α-subunit and β-subunit. Based on the β-subunit, inhibin can be of two types, inhibin A (βA subunit) and inhibin B (βB-subunit). Inhibin B is more biologically active compared to inhibin A. They are secreted from granulosa cells of the ovary and Sertoli cells of the testes. Inhibin is also synthesised from the placenta and can be present in the foetus. Unlike activin, it inhibits the synthesis and release of the FSH. Apart from FSH inhibition, inhibin exerts paracrine effects on the gonads. Inhibin functions as a regulatory hormone in mares during the follicular phase of estrous cycle. It inhibits progesterone synthesis in the ovary. The concentration of inhibin decreases with a declining ovarian follicular reservoir; hence, it can be used as a potential marker for ovarian function. Inhibin is three times more potent than follistatin.

22.1.1.3.5.3 Relaxin

The predominant source of relaxin is the corpus luteum in both pregnant and non-pregnant animals. It is also synthesised in the placenta (horse). In males, relaxin is pro­duced from the prostate and released in seminal fluid. In recent years, the heart’s atria have been identified as an extragonadal source of relaxin. The functions of relaxin include inhibition of uterine contractility and relaxation of the uterine muscles and ligaments during pregnancy in syn­ergy with progesterone, inhibition of the collagen synthesis in the estrogen-primed cells of the cervix, vagina, and pubic symphysis to soften the birth canal. It promotes angiogenesis in the endometrium, especially during the implantation of mares. In horses and rats, relaxin has an important role in ovulation to guiding the ova into the fallopian tube. It causes proteolysis in the follicular walls by influencing the secretion of gelatinases and tissue inhibitors of metalloproteinases. The receptors for relaxin are present in granulosa and theca cells of follicles; thus, it facilitates follicular development in pigs and humans. It is also involved in mammogenesis.

22.1.1.3.5.4 Follistatin

Follistatin is a single chain glycoprotein of the ovarian follic­ular fluid that acts as an activin-binding protein. It neutralises activin and indirectly suppresses FSH secretion from the anterior pituitary. Follistatin can be neutralised by activin.

22.1.2 Role of HPO Axis in Female Reproduction

The events of the female reproductive biology are regulated by a complex interplay between the nervous and endocrine systems. The hypothalamus, the central component of the HPO axis, secrets GnRH to stimulate pituitary gonadotrophs for secreting FSH and LH. The gonadotropins (FSH, LH), in turn, regulate the gonadal functions. Together with inhibins and activins, ovarian steroids influence gonadotropin secre­tion in a feedback manner. The FSH acts on the ovarian follicle’s granulosa cells and controls folliculogenesis and estrogen synthesis. A high level of estrogen, low level of LH, and absence of inhibin initiate follicular recruitment. The follicular dominance is achieved in a milieu with low FSH and high LH and inhibin. Inhibin suppresses the FSH secre­tion from the pituitary and restricts further follicular growth. The estrogen is produced from the matured follicles under the influence of FSH and LH (details in two cell two gonadotro­pin mechanism). When the estrogen level reaches a thresh­old, it helps to manifest the behavioural estrus. It stimulates the GnRH surge centre at the pre-optic and suprachiasmatic centre of the hypothalamus to release GnRH at high pulses, which facilitates pre-ovulatory LH surge and ovulation.

The theca and granulosa cells undergo luteinisation after ovulation, and the corpus luteum is formed. The large luteal tissue secrets progesterone and oxytocin, whereas the small luteal tissue secrets progesterone. The progesterone supports pregnancy by causing endometrial hypertrophy, secretion of uterine milk and blocking uterine contraction (details in the endocrine control of pregnancy). A higher progesterone level exerts strong negative feedback over the hypothalamus and prevents pre-ovulatory follicular growth. If an animal fails to conceive, the corpus luteum undergoes luteolysis by the action of PGF2α. The PGF2α is secreted from the endome­trium by the action of oxytocin. After binding with its recep­tor at the endometrium, oxytocin stimulates the enzymes for the synthesis of PGF2α from cholesterol. Estrogens upregulate the expression of oxytocin receptors in the uterus. The synergistic and agonistic activities of the gonadotropins and sex steroids to control the HPO axis have been summarised in Table 22.3 and Fig. 22.1.

22.1.2.1 Factors Affecting the HPO Axis

Several factors regulate the HPO axis.

Table 22.3 Interrelationship between the gonadotropins and sex steroids

22.1.2.1.1 Genetical and Congenital Factors

Some animals may have inherited HPO axis insufficiency due to genetic mutations of the HPO axis components. Turner syndrome, Kallmann’s syndrome (in humans) and chromo­somal aberrations may directly affect the HPO axis. Hypothalamic-pituitary signal dysfunctions may occur due to genetic disorders that cause acute ischemia or compression and autoimmunity.

22.1.2.1.2 Pathological or Physiological Dysfunction Obesity, hyperprolactinemia and hypothyroidism affect the HPO axis by reducing GnRH and gonadotropins. The extragonadal sources of estrogens may occasionally disturb the ovarian function in conditions like Cushing’s syndrome, tumours or cysts in the adrenal or ovary and congenital adrenal hyperplasia. In these conditions, conversion of peripheral androstenedione to estrone in adipose tissue and skin is increased, and this estrogen causes cyclic irregularities. This phenomenon is common in polycystic ovarian syndrome. Chronic parasitism causes prolonged hypothyroidism, leading to reduced GH-dependent IGF-I synthesis in the liver, resulting in inhibition of the HPO axis. Due to regional acute ischemia or compression and autoimmunity, panhypopituitarism also affects the HPO axis. Phytoestrogenic compounds negatively affect the HPO axis. Metabolic disturbances, such as ketosis in high yielding cows, reduce the secretion of both FSH and LH. The metabolites like high GH, non-esterified fatty acid (NEFA) and beta-hydroxybutyrate (BHB), and lower glucose and IGF-I are thought to modulate the HPO axis under these conditions.

22.1.2.1.3 Stress

Stress-induced suppression of the HPO axis occurs under the influence of gonadotropin-inhibitory hormone (GnIH) and cortical hormones (Fig. 22.4). The GnIH is also called RFamide-related peptide 3 (RFRP) in mammals. Its concen­tration is increased during stress and leads to inhibition of GnRH secretion. Synthesis of cortical-releasing factor (CRF) during stress reduces GnRH secretion as both CRH and GnRH neurones are localised in the pre-optic area at the

Fig. 22.4 Factors control the release of gonadotropins. The sketch shows various factors that are controlled the release of gonadotropins, the LH (luteotropic hormone) and FSH (follicle-stimulating hormone) from the KNDy (kisspeptin-neurokinin B-dynorphin) Neuron or neuronal network and GnRH (gonadotropin) Neuron of the hypothala­mus followed by the pituitary gland. The network systems are depicted by bold circles, bold arrows and circled positive (+) sign. Dotted circles and bold arrows illustrate the negative or inhibitory factors and a circled negative (-) sign. Most factors are affected by the KNDy Neuron, viz. GnIH (gonadotropin inhibitory hormone), an RFamide-related peptide 3 (RFRP3); various sex steroids in variable concentrations (e.g. E2 = estrogens, P4 = progesterone and T = testosterone); in the stress-causing release of cortisol, hypoglycaemia and similar conditions; occurrence of physiological dysfunctions like cyst, acute and chronic illness; disturbances in metabolism due to leptin defi­ciency, hyperglycaemia, and other conditions; and various drugs like prolactin inducer or stimulating, opioids, etc. Among such factors, GnIH can also directly affect GnRH neurons and the pituitary gland. The sex steroids, viz. E2 and T in various concentrations can directly affect LH and FSH’s functional activity

hypothalamus. CRF down-regulates GnRH gene expression in the hypothalamus (Fig. 22.5). FSH secretion is more affected in response to stress than LH secretion. In nutritional and environmental stress, HPO axis is affected through

Fig. 22.5 Effect of stress on the female reproductive system. Figure shows the stress caused by to release of CRF (cortical-releasing factor) over the hypothalamus, followed by the pituitary gland and female reproductive system. It affects GnRH (gonadotropin-releasing hormone) and GnIH (gonadotropin-inhibitory hormone) neurons in the hypothalamus. Receptors for GnRH, LH (luteinising hormone) and glucocorticoid hormone in the pituitary gland are affected as a conse­quence of it. Ultimately, estrogen production and follicular development have been arrested. The down arrow denotes the various modulations of the mechanism of actions for inhibitory action, the upward arrow by influencing action and both side arrows indicate the together actions

specific mediators like adipokines, cytokines, and adipose tissue-derived factors, fatty acids and (Table 22.4). Long­term exposure to corticosteroids, ACTH and stress, cause an inhibitory effect on the HPO axis. But, acute release or administration of ACTH and some corticosteroids to the estrogen-primed animals cause stimulation of the HPO axis and pre-ovulatory LH surge. Malnutrition causes decreased adipose tissue-derived leptin levels, and the stomach originates ghrelin. These two peptides result in a decrease in GnRH and LH pulse.

22.1.2.1.4 Environment

Season and photoperiod affect the HPO axis, particularly in the temperate region, through melatonin production in the pineal gland (discussed in detail in the puberty section). Seasonal influence on the reproductive cycles in seasonal breeders also operated through the action of melatonin over the HPO axis. The HPO axis is affected in cattle when the temperature-humidity index (THI) exceeds 70, and high THI affects the feed intake, growth, and hormonal imbalances to suppress the HPO axis.

22.1.2.1.5 Physiological Factors

Inter-estrus interval is variable and can be modulated in different species due to genetic variation and in the animals’ specific physiological state like the postpartum period. High

Table 22.4 Effects of various adipokines, cytokines and adipose tissue-derived factors, fatty acids and peptides on HPO axis

Adipokines

Cytokines

Fatty acids

Peptides

Source: Calejja-Agius et al. (2009), Prins et al. (2012), Tsatsanis et al. (2015), Dobrzyn et al. (2018), Hu et al. (2019)

GnRH gonadotropin-releasing hormone, LH luteinising hormone, FSH follicle-stimulating hormone, E2 estrogens, P4 progesterone, IGF insulin­like growth factor, PCOS poly cystic ovarian syndrome, MIF Mullerian inhibiting factor, CL corpus luteum, DMN dorsomedial hypothalamic nucleus, POA pre-optic area, AVPV periventricular nucleus, INF infundibulum, ARC arcuate nucleus

yielding cows undergo ketosis immediately after the parturi­tion due to high GH, non-esterified fatty acid (NEFA), beta­hydroxybutyrate (BHB) and low insulin, glucose, and IGF-I. This condition reduces the secretion of both FSH and LH.

In the postpartum period, high metabolic disturbances (ketosis in high yielding cows) occur. All the factors that affect the puberty and estrous cycle are mediated through the various components of the HPO axis.

22.1.2.1.4 EndocrineFactors

22.1.2.1.6.1 Prolactin

Estrogens influence the lactotroph cells of the anterior pitui­tary to secrete prolactin during puberty and late pregnancy. Prolactin inhibits gonadotropin secretion. In birds, prolactin plays a significant role in brooding behaviour and is associated with metabolic changes that occur during brooding.

22.1.2.1.6.2 Oxytocin

Oxytocin, a hypothalamic neuropeptide stored in the poste­rior pituitary, stimulates the uterine smooth muscle activity. It acts synergistically with estrogens during parturition to make strong myometrial contractions.

22.1.2.1.6.3 PGE2

PGE2 produces in the ovary, uterus, and embryonic membranes. It helps to soften the cervix, augments uterine contraction, and prepares the tract during parturition, particu­larly in horses and humans. It is also involved in ovulation and progesterone secretion from the corpus luteum.

22.1.2.1.6.4 Human Chorionic Gonadotropin (hCG)

The hCG is a glycoprotein synthesised in the trophoblast cells of a blastocyst in primates and humans. Its function is similar to LH, so it helps to secrete progesterone and estrogens. It can protect the embryo from the maternal immune system during the first phase of pregnancy. To confirm pregnancy, the presence of this hormone can use in the maternal blood.

22.1.2.1.6.5 Equine Chorionic Gonadotropin (eCG)

The eCG, a glycoprotein, is released from the chorionic tissues of the placenta in horses and primates. It acts like FSH and helps with follicular growth and ovulation. Thus, it helps form accessory corpora lutea to ensure progesterone production during pregnancy. The eCG uses to induce super­ovulation in other species.

22.1.2.1.6.6 Placental Lactogen

Placental lactogen is a polypeptide hormone of placental origin. It has structural and functional similarities with growth hormones. Hence, it is called chorionic somatomammotropin. It helps to provide energy to the devel­oping foetus during pregnancy by altering the metabolic status of the mother in humans. It has an important role in lactation and maintenance of corpus luteum in the rat. Pla­cental lactogen supports the pregnancy by the steady proges­terone production from the corpus luteum. There are two forms of placental lactogen-I and II. The placental lactogen- I can bind with the same receptor as prolactin due to struc­tural similarity and can mimic the activity of prolactin.

22.1.2.1.6.7 Summary of Endocrine Factors Involved

in Female Reproduction

According to the involvement of female reproduction, repro­ductive hormones can be classified into three types. These are primary, secondary, and tertiary hormones (Table 22.5). Pri­mary reproductive hormones have a direct role in reproduction and secondary hormones are involved indirectly by influencing the growth and functional activity of repro­ductive organs or related endocrine glands. Tertiary hormones are the neurohormones that engage in the secretion of other reproductive hormones.

22.1.3 Pheromonesand Pheromone-Induced Sexual Behaviour

The chemical substance synthesised and released by the animals into the surroundings affects the physiology and behaviour of the other animals of the same species called pheromone. Pheromones are an important medium of com­munication in both mammals and non-mammalian species. Pheromones play crucial roles in searching for mates, searching for foods and other interactions like alarming predators. Pheromones can be either male-specific, female­specific, or combined types. In mammals, pheromones are released through urine, faeces, vaginal secretion, saliva, and modified scent (cutaneous) glands, including hair and wool. The receiving animals perceive pheromones through their olfactory system or in combination with other sensory systems (auditory, visual, or tactile system) and combine with odorant-binding proteins (OBPs), major urinary proteins (MUPs), and/or similar soluble pheromone carrier proteins for their transport through biological fluids. The MUPs are produced in the liver and excreted through urine. The OBPs are mostly found in nasal tissues. The 1-octen-3-ol is one of the soluble pheromone carrier proteins. It also increases the bioavailability of the pheromones by protecting them from metabolism. Sex pheromones are exclusively involved in the socio-sexual communications between the male-female interactions (Table 22.6). Sex pheromone modulates acceler­ation of puberty, induction of estrus, minimises the postpar­tum anestrus, exhibition of estrus symptoms, synchronisation of ovulation, influences courtship and maternal behaviour, including reduced aggressiveness to young by influencing synchronise hormonal activities (Table 22.7). Sex pheromones are of three types. (a) Releaser pheromones— Releaser pheromones are the pheromones which cause imme­diate response like immobilisation reflex in the sow. (b) Signaller pheromones—Signaller pheromones indicate the identity or presence of the pheromone producer (sender) like a mother-neonatal relationship. (c) Primer pheromones—Primer pheromones make slow and long- acting responses like the advancement of puberty and reduc­tion of postpartum anestrus duration.

22.1.3.1 Bio-stimulation (Interaction with the Opposite Sex)

The stimulus induced by the presence of males to modulate estrus and ovulation through pheromones, genital

Table 22.5 Hormones and growth factors involved in female reproduction with their sources

stimulation, or other external cues (including olfactory, visual, and auditory signals) is called bio-stimulation. Differ­ent animals exhibit sex behaviours through bio-stimulation.

22.1.3.1.1 Flehmen Response (Flehmen Reaction or Flehming or Flehmening)

The flehmen response is a typical behavioural posture of the animal after receiving the pheromone from the environment. The behavioural posture includes twisting the upper lip to expose the front teeth and gum with the extension of the head for better inhalation through the nostril with deep breathing for a few seconds. This response is mainly exhibited by the males, often in females, of various ungulate mammals, including cattle, buffalo, sheep, goats, horses, and cats (like a domestic cat, tiger, etc.). It is also displayed by rodents like guinea pigs and mice. The chemicals (pheromone) or scents that the animals perceive are non-volatile organic compounds (non-VOCs) and secreted mainly through the urine and or genital organs of the females during proestrus and estrus, the time of sexual receptivity. The perceptive organs of these pheromones are the vomeronasal organ (VNO) for the pheromones in liquid form and main olfactory system (MOS) for aerosol molecules. During the time of inspiration, the lumen of VNO increases and blood pressure decreases to facilitate proper mixing of pheromones with mucus-binding protein, followed by increases in blood pressure and decreases in VNO lumen, which expel the pheromones from the VNO organ. In buffalo, rapid tongue strokes at the rostral and medial palate generate a separate route from the prompt response. This response attracts the male for

Table 22.6 Source and chemical nature of some sex pheromones in domestic animals

Cat

Source: Wani et al. (2013) and Mucignat-Caretta (2014)

Table 22.7 Role of some sex pheromones in different species

courtship and improves the sperm quantity and libido in males. The characteristics of Flehmen response are used to identify the estrus in females to synchronise reproduction and breeding management in wild ungulates. Animals also exhibit flehmen response after parturition to recognise the neonates, where females perceived the non-VOCs from the newborn and the amniotic fluids. It often occurs in sheep, horses, and elephants. Goat shows the flehmen reaction in response to the pheromone synthesised from the modified sex hormone (androgen) and excreted through urine.

22.1.3.1.2 RamEffect

The presence of ram in the flock of ewe can promote the occurrence of estrus. This phenomenon is called the ram effect. It is widely practised in sheep farming to synchronise the estrus and reduce the silent estrus incident. Introduction of the ram for at least 9-13 days before the end of the anestrus augments ovarian activity and fertility. Bio-stimulation for the ram effect mediates through the pheromones secreted through the wool and wax of the ram (Table 22.6), which induces the LH secretion in anestrus ewes. The characteristic behavioural response of the ram effect includes cessation of movements of ewes during estrus after the physical contact with the rams. This typical sheep behaviour is called tupping and is used to detect the estrus in ewes.

Besides the ram effect, pheromone-induced behavioural responses are manifested by the ewes for offspring recogni­tion, the development of filial attachment in ewes, and the development of suckling behaviour in lambs. The maternal- offspring passion also develops in response to the pheromones in amniotic fluid other than oxytocin secreted during parturition. The pheromones also assist in developing the perpetual olfactory memory in the brain.

22.1.3.1.3 Self-Enurination (SE) in Buck (Scent-Urination, Urine-Marking)

Domesticated bucks show a typical behavioural response during their breeding season termed SE. The spreading of urine characterises it onto the face, beard, and front legs to display olfactory signals to attract estrus females. The buck turns its head and shoulders downwards and emits urine from the erect penis to disperse the urine onto the face and beards. The manifestation of SE occurs when a buck approaches estrus females before mating to induce estrus and ovulation. The vocalisation of bucks also causes a similar effect.

22.1.3.1.4 BullEffect

The resumption of postpartum ovarian activity in cows is seen when the bulls (even vasectomised) are introduced to the farm. This phenomenon is called the bull effect. The onset of puberty in cows can accelerate through bio-stimulation. The cervical mucus of estrus females can promote ovarian activity in heifers and postpartum cows. The pheromones secreted from cattle are amended in Table 22.6. The pheromones such as 1-iodo undecane, secreted through urine and faeces of females during estrus, can induce increased libido in bulls. Hence, the 1-iodo undecane is considered a biochemical marker to identify bovine estrus.

22.1.3.1.5 Vandenbergh Effect

John Vandenbergh first described the sexual behavioural response due to bio-stimulation in mice. He reported that puberty could be accelerated in female mice exposed to the urine of adult males. Vandenbergh effect is also seen in pigs. Boar pheromones (Table 22.6) are responsible for the Vandenbergh effect in females to stimulate reproductive behaviour and performance in gilts and sows. The male pheromones (5α-androsterone-16-en-3-one and 5- α-androsterone-16-en-3α-ol) are produced in testes and sub­maxillary salivary glands that are released through saliva. The sow comes close to the boar and makes the nose to nose contact with frequent movement of the tongue to receive the pheromones through VNO. This posture of sows is called chomping behaviour. The pheromone causes immobilise (or standing) reflex in estrus sow to hasten the copulation process. The saliva of boars is often used to augment repro­ductive activity in females. A group of fatty acids (Table 22.6) secreted from the skin of lactating sow reduce the agonistic behaviour in piglets for feed and house space after weaning.

22.1.3.1.6 Pheromone-Induced Sexual Behaviour

in Dog and Cat

Pheromones secreted from sebaceous glands of male dogs and cats enhance the sexual in females. The supra-caudal glands in the peri-anal region of the cat and circumanal glands of the dog around the anus become active during spermatogenesis and estrus, respectively, to promote court­ship behaviour in both the sexes. Methyl-dihydroxy benzoate secreted from the sebaceous gland of bitches during estrus can modulate sexual behaviour in males. A significant quan­tity of pheromones secretes in cats (Table 22.6) with specific functions. The tomcats rub their face to deposit facial pheromones (F2 type) in an object near sexually active females as a sexual display that improves courtship.

22.1.3.1.7 WhittenEffect

In mice, the presence of a male in a group of females can initiate ovarian activity and stimulate gonadotropin secretion. This phenomenon is called the Whitten effect. The phero­mone of male mice is secreted through urine, and the females perceive it through smell. It causes synchronisation of estrus in females with irregular sexual cycles. The pheromones responsible for the Whitten effect are 2-sec-butyl- dihydrothiazole (SBT) and dehydro-exo-brevicomin (DHB). The same compounds also cause puberty augmentation in mice called the Vandenbergh effect.

22.1.3.1.8 BruceEffect

The presence of an unknown male terminates the pregnancy in female rodents called the Bruce effect. It was named after Hilda Margaret Bruce, who identified the behaviour in 1959. The major histocompatibility complex (MHC) class 1 protein released from the males through their urine is perceived through VNO. The females learn the specific MHC of the males during mating. Still, when an unfamiliar male is introduced into the pregnant flock, the scent released by the male activates the neuro-endocrine pathway via the cortico- medial amygdala, accessory olfactory tract, and stria terminalis, which in turn stimulates the release of dopamine. Dopamine prevents the secretion of prolactin and causes pregnancy termination. The Bruce effect can also be seen in pigs and domestic horses. The mouse can sense estrogen through nasal ingestion; hence, excess estrogen in pen may cause the Bruce effect. The female rodents perceive MHC molecules through VNO. Oxytocin helps to recognise the MCH molecules of the breeding partner and does not termi­nate the pregnancy.

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Musk

The organic aromatic substance secreted from the preputial gland of a kind of male deer (Moschus spp.) inhabiting China, India, Pakistan, Afghanistan, Tibet, Siberia, Mongolia, and North Vietnam is termed ‘Musk’. The deer is named musk deer. Muscone is the main active ingredient having several androstane derivatives with specific proteins. The deer secrets musk to define the territory and attract females. Unmated male musk deer produce a more significant amount of muskcone than mated males due to certain bacteria in higher concentrations that control the meta­bolic pathways of androstane derivatives. Certain plants and animals produce similar kinds of strong odorous substances called musk plants and musk animals (muskrat, musk duck, musk turtle). Musk is one of the most expensive animal products commer­cially used as a perfume fixative in the world.

22.1.3.1.9 Interspecies Communication

Pheromones secreted from the urine, vaginal fluid, saliva, faeces, and milk during different phases of the cow’s estrous cycle can attract other species like dogs, mice, and rats. These animals can identify estrus cows, and dogs can sense the luteal phase of cows.

22.1.3.2 PheromonelikeChemicals

Some bio-chemicals produce by one animal influence the activity of others, but effects are not similar to pheromones like allomones, kairomones, synomones, and interomones. These are generally released from insects. Allomones are the semiochemicals released by one organism to affect the behaviour of the other species to favour the sender, not the receiver. Many insects (stink bugs, bombardier beetles, blis­ter beetles) use the allomones to defend the predators by emitting pungent smells or repugnant chemicals. Kairamones are the semiochemicals that benefit the receiver, not the sender. Kairamones are used by parasites, predators, parasitoids, omnivores, and herbivores to search for food. Synomones regulate interspecific communications benefitting both the sender and the receiver and can control both intraspecies, and some are interspecies communications. Interomones are semiochemicals which affect the physiolog­ical response of other species.

22.1.3.2.1 Endocrine Disruptors

The chemicals that interfere with the functions of the endo­crine system by either mimicking the hormonal activity or acting as anti-hormone are called endocrine disruptors (EDs) or endocrine-disrupting chemicals (EDCs). They can gener­ate inside the body due to disturbance in the synthesis process or can introduce from the environment as synthetic chemicals. The synthetic EDs that affect reproductive functions are diethylstilbestrol (DES) or stilbestrol (nonste­roidal estrogen); industrial chemicals like phthalates, bisphenol A; pesticides (DDT) and organochlorine pollutants like polychlorinated biphenyl (PCBs). They cause DNA methylation or reprogramming of the genes in germ cells and affect the follicular dynamics. Long-term exposure to phytoestrogens like clover and fungal toxin contaminated feed cause reproductive disturbances in sheep.

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EDCs and Infertility

Exposure to endocrine-disrupting chemicals (EDCs) and persistent organic pollutants (POPs) causes infertility in the animals. Female mammals are more vulnerable to EDCs and POPs, which can be transmitted through the placenta and milk and may cause congenital anomalies in the endocrine system. The wild animals are also suscep­tible to EDCs and POPs-induced infertility through the milk and food web. The animals of the polar regions of the globe are at higher risk due to more concentrations of the POPs in those regions.

22.2