Fertilisation
Fusing the pronuclei of the male and female gametes and intermingling the chromosomes of maternal and paternal origin to form a diploid zygote is known as fertilisation. In mammals, the fertilisation occurs inside the body of females and is hence called internal fertilisation.
The introduction of sperm into a female’s reproductive system is called insemination, which can be done naturally and artificially. In natural mating, the semen is introduced into the vagina or close to the cervix, whereas during artificial insemination, semen is mostly deposited into the cervix or uterus. Soon after the semen deposition, the sperm has to travel through the cervix towards the uterus to reach the fallopian tubes, where it interacts with the ovum. In most mammals, the fertilisation usually occurs in the oviduct’s ampullary-isthmus junction. The period between insemination and sperm-ovum interaction is crucial for fertilisation as both sperm and ovum have a fixed fertilisable life span within which they have to interact (Table 23.1). The sperm has to reside in the female reproductive tract for a considerable period to achieve maximum fertility, during which the spermatozoa undergo some changes to become fertile. This process is called capacitation and the time required for capacitation varies between species (Table 23.1). The fertilisation process is a series of speciesspecific biochemical and molecular changes that include receptor-ligand interactions, activation of signalling cascades and nuclear transformations. 23.1.1 SpermTransportation
The movement of sperm in the female genital tract from the site of semen deposition to the site of fertilisation (ampulla) is called sperm transportation. The transport of spermatozoa in the female reproductive tract occurs in three phases: initial rapid transport, colonisation and sustained transport.
During natural copulation, the seminal fluid is usually deposited in the anterior vagina. Then the spermatozoa are colonised inside the crypts of the cervix and released in two modes. In the first mode, the spermatozoa exhibit initial rapid transport and reach the distal end within 15-30 min in most species (Table 23.1). Still, most of these spermatozoa cannot fertilise the ovum as the capacitation is not completed. The initial rapid transport is facilitated by the contractility of the female reproductive tract rather than sperm motility. In the second mode of transportation, the maximum spermatozoa undergo slow transport by swimming through the cervical mucus. Sperm transport depends upon the structure and activity of cervical, uterine and fallopian tube epithelial cells and the contractile activity of smooth muscles of these organs. Ovarian hormones mainly control the epithelium’s secretory activity, favouring sperm transport, whereas progesterone suppresses it. The contraction of the uterus and fallopian tube is controlled by oxytocin released during intercourse. The contractility of the uterus and fallopian tubes is also affected by prostaglandins. Table 23.1 Preparatory time for the gametes for fertilisation in female genitalia
| Species | Site of inseminationa | Time for initial rapid transport (min) | Time for sperm capacitation (h) | Time for ova migration/ maturation (h) |
| Cattle | Cervix (vagina) | 2-15 | 2-4 (6 max) | 8-10 (72 max) |
| Buffalo | Cervix (vagina) | 5-15 | 4-8 | >4 |
| Sheep | Cervix (vagina) | 6-10 | 1-2 | 10-25 (66 max) |
| Goat | Cervix (vagina) | 10-15 | 4 | 98 (max) |
| Horse | Uterus (cervix or uterus) | 15-30 | 3-24 | 30-36b (144 max) |
| Pig | Uterus (cervix or, uterus) | 15-30 | 2-6 | 12-48 |
| Dog | Uterus (uterus) | Several minutes | 2-4 | 4-7 (days)c |
| D. cat | Vagina or uterus (vagina or cervix) | 20 | 3-4 | 18-36 (144-168 max) |
| G. pig | Peritoneum (uterus) | 1-30 | 16-18 | 5-10 min (48 max) |
| Rat | Uterine horn (uterus) | 15-30 | 1-4 | 88 (max) |
| Rabbit | Vagina (vagina) | Few minutes | 5-11 | 55 (max) |
| Mice | Vagina (vagina) | 15 | 2 | 72 (max) |
| Human | Vagina (vagina) | 5-30 | 5-7 | 24 (80 max) |
D. cat Domestic cat, G. pig Guinea pig, max maximum
a Insemination considered as artificial insemination; and the site of natural insemination mentioned in the bracket b In in vitro
c Longer duration is required due to completion of meiosis-I and II (up to metaphase-II)
23.1.1.1 Site of Semen Deposition
The site of semen deposition varies considerably among species and the mode of insemination (Table 23.1). The semen is deposited in the anterior vagina during natural mating in most domestic animals. Some species, such as pigs and horses, deposit the semen directly into the uterus. The murine rodents deposit semen directly into the uterus, but some remain in the vagina and coagulate to form a copulatory plug promoting sperm transport. Coagulation also occurs in human semen, but it forms a loose gel, unlike murine rodents. The gel’s structural proteins comprise semenogelin I and semenogelin II, and their glycosylated form is secreted from seminal vesicles.
The gel is degraded by prostate-specific antigen (PSA). The intention of forming the gel is to hold the spermatozoa at the cervical os and to give protection from the vaginal environment. The semen of some primates also coagulate to form a soft gel. Female chimpanzee mates with more than one male, and a compact gel produced from the semen prevents further mating. The copulatory tie forms during the mating in some carnivores where the penis serves as a copulatory plug for sustained insemination. 23.1.1.2 Transportation Through Vagina
The spermatozoa have to overcome the immune defence machinery of the vagina that acts as a natural barrier against external infections. These include acidic pH (pH 4) of the vagina and other immunological responses. The acidic pH of the vaginal secretion is due to the lactic acid produced by the action of anaerobic lactobacilli over the vaginal epithelium from glycogen. The acidic vaginal pH is detrimental to spermatozoa. The alkaline seminal plasma neutralises the acidic vaginal environment. The secretions of seminal vesicles, prostate gland and bulbourethral glands are alkaline (details in accessory sex gland section), which neutralises the vaginal acidic medium (pH 7.2) within a few minutes of post insemination and maintains this pH for up to 2 h. The spermatozoa become more motile in an alkaline medium. The immunosuppressive agents present in the seminal plasma protect the spermatozoa from the immune protective components of vaginal secretions. The immunosuppressive agents of the seminal plasma include prostasomes, prostaglandin E, polyamines, immunoregulatory cytokines and lymphocyte suppressing proteins. Immediately after the insemination, the semen coagulates in the acidic medium to form a clot. The proteolytic substances of the seminal plasma dissolve the clots and allow the spermatozoa to move within 30 min. Spermatozoa that cannot move within 2 h remain in the vagina even up to 12 h, depending on species, but almost lose their motility.
At first, a rapid sperm transport is occurred within seconds after ejaculation, followed by slow transport. The majority of spermatozoa move through a slow transport process. 23.1.1.3 Transportation Through Cervix
The spermatozoa reach the internal os and cervix within 1.5-3 min of insemination by sperm motility and contractility of the myometrium. In most domestic animals, the spermatozoa are trapped within the mucosal folds of the cervical crypts that act as sperm reservoirs. The spermatozoa are released slowly by the female reproductive tract’s motility and contractile activity. The cervix provides a favourable environment to spermatozoa and protects them from phagocytosis. Cervix also aids the sperm energy metabolism and helps the capacitation process. Cervix also helps in sperm selection and restricts the entry of non-motile and abnormal spermatozoa. The cervix allows the sperm migration nearer to ovulation time and blocks the sperm entry during other phases of the reproductive cycle by forming a cervical plug.
The cervical mucous acts as a barrier to sperm transport. The cervical mucous comprises flexible linear glycoprotein molecules named mucins. The long mucin molecules are aligned themselves to form a hydrogel of the 3D network. The mucin molecules are glycosylated during the oestrus under the influence of oestrogen. The glycosylated mucins have a water holding capacity, and the mucous become highly hydrated during oestrus. The cervical plug liquefies during oestrus under oestrogen and highly hydrated cervical mucous, and the cervix appears to widen to allow the sperm to move through the cervix. The spermatozoa penetrate the cervical mucous through their motility and rheological properties. The spermatozoa swim in a straighter path guided by the secretory flow of cervical mucous with the orientation along the long axis of mucin threads. The non-motile spermatozoa are unable to penetrate the cervical mucous. Thus, cervix helps to select the non-motile spermatozoa.
The penetration of cervical mucous by the spermatozoa is facilitated by a glycoprotein called beta-defensin 126 (DEFB126) identified in primates. The DEFB126 provides a highly negative surface charge to the spermatozoa essential for cervical mucous penetration. The sperm has to escape the immune responses of the cervix as the insemination stimulates the migration of neutrophils and macrophages into the cervix. The normal and motile spermatozoa can avoid phagocytosis, but neutrophils phagocytose the non-motile and abnormal spermatozoa due to complementfixing anti-sperm antibodies. The neutrophils interact with the spermatozoa through L-selectin that binds with the sialic acid on the sperm surface. Human sperm moves at a speed of 1.5-5 mm/min in the female genital tract and reach the uterus within an hour. The pig, horse and dog deposit the semen directly to the uterus or end of the cervix; hence, the spermatozoa cross the cervix or uterus rapidly. 23.1.1.4 Transportation Through Uterus
Contractile activity of the uterine smooth muscles along the length of the uterus propels the spermatozoa and watery cervical mucus from the cervix into the uterus. Strong contractile activity of myometrium is seen during oestrus compared to the luteal phase in cows and ewes. The contraction of uterine smooth muscle is stimulated by oestrogen that stimulates the secretion of PGF2α. The oestrogen from the pre-ovulatory follicles reaches the endometrium through a counter-current exchange mechanism between the ovarian vein and ovarian artery, then transported through the uterine artery to act over the endometrium. In boars, seminal plasma contains a considerable amount of oestrogens directly deposited into the uterine cavity and aids the uterine contraction. In certain animals, like cow and rabbit, mating or copulation induce uterine contraction probably by releasing oxytocin. Stimulation of vulva and per-rectum uterine messages during artificial insemination may have a similar effect. Fright or fear may inhibit sperm transportation due to the release of epinephrine causes vasoconstriction.
23.1.1.5 Transportation Through Utero-Tubal Junction (UTJ)
The UTJ acts as an anatomical and physiological barrier to sperm from the uterus to the oviduct. This UTJ barrier resembles a filter that allows only healthy motile sperm to the isthmus. The abnormal and poor-quality sperm are arrested here. The structure of UTJ is simple in humans, but in cows, pigs, rabbits and many other species, the structure is complicated due to numerous mucosal folds. Cul-de-sacs characterise the bovine UTJ originated from the mucosal folds that entrap the spermatozoa. The lumen of UTJ is squeezed during the oestrus by the contraction of thick, smooth muscle. The presence of viscous mucus in the lumen of UTJ further restricts the passage of sperm. The spermatozoa penetrate the utero-tubal junction by their linear progressive motility. After crossing the UTJ, the viable spermatozoa reside in the isthmus, attaching to its wall for capacitation. Different proteins, namely fertilin β, calmegin or testis-specific angiotensin-converting enzyme (ACE), help sperm migration through UTJ. Infertility may result due to deficiency of these proteins.
23.1.1.6 Sperm Transport in the Oviduct
From the millions of ejaculated spermatozoa, nearly 10,000 or fewer spermatozoa enter the fallopian tube. The spermatozoa can enter any side of the tubes, but those that enter ipsilaterally to the ovulation side are capable of fertilisation. The oviduct provides a favourable environment for the spermatozoa as the oviduct does not induce any immune reactions, unlike the vagina, cervix and uterus. The isthmus is considered a sperm reservoir in cattle, sheep, pigs, rabbits, mice and other species. In addition to the cervix and UTJ, the oviduct itself acts as a barrier in sperm selection. Only viable spermatozoa bind with the wall of the fallopian tube epithelium till the fertilisation and rest are eliminated. The interactions of sperm with oviductal epithelial cells (OEC) are mediated through receptor-ligand interactions, and this binding enables the sperms to become viable for at least 48 h. The bovine seminal vesicle proteins like PDC109 (seminal plasma A1orA2, Binder of SPerm (BSP), viz. BSPA3 and BSP30K present in the sperm heads interact with the annexin family of proteins of OEC. The BSP also contains two heparin-binding domains. BSP’s binding with heparin blocks BSP-annexin interaction and detachment of spermatozoa from the oviductal epithelium. Hence, heparin is extensively used in in vitro capacitation process. Two proteins, namely Hsp60 and GRP78 of the apical membrane of the bovine and human OEC, are capable of binding with the sperm membrane.
23.1.1.7 Sperm Loss During Transportation and Its Manipulation
The numbers of spermatozoa are gradually reduced during the transportation from the deposition site to the site of fertilisation. In sheep, its number reduces from 1000 million to only 1000 from the site of deposition to the site of fertilisation, respectively, in pigs the number varies from 8000 million to 1000, in rats 60 million to 20-100, in guinea pig 80 million to 25-50, and in humans 200 million to 10-1000. The insufficient sperm numbers at the site of fertilisation lead to fertilisation failure, particularly in sheep and pigs.
The sperms are susceptible to phagocytic attack by the neutrophils and macrophages infiltrated into the uterine lumen after coitus. This process is called spermophagy and is mostly seen in mice, rats and rabbits. The damaged sperms and seminal debris are mainly vulnerable to phagocytic attack, and seminal plasma constituents generally protect the normal sperms. But the normal sperms are deposited into the anterior vagina and are also susceptible to phagocytic attack as their immune protections are when they reach into the uterus. In dogs, endometrial glands act as sperm reservoirs. In some species with an extensive ovarian bursa (mice), the non-fertilised sperm can enter the peritoneal cavity.
The induction of oestrus by progestogen or prostaglandin F2α sometimes causes disturbances in sperm transport in the ewe. Prolonged exposure to phytoestrogen and abrupt luteolysis may disturb sperm transportation at the subsequent oestrus. All these disturbances in sperm transport are due to a lack of optimum oestrogen level. The sperm transportation can be enhanced by adding some compounds to the semen or applying them to the female reproductive tract. Majorities of these compounds are species-specific such as prostaglandin E1 in rabbits, sheep and goats, estradiol-17β in sheep, carbacholine in pigs and amylase or glucuronidase in cattle.
The spermatozoa undergo two important pre-fertilisation events for its activation at the isthmus: the capacitation and hyperactivation.
23.1.2 Capacitation
The capacitation is the physio-biochemical alterations in the sperm plasma membrane that enables them to become fertile (Table 23.2). The site and time required for capacitation vary between species (Table 23.1). In species where sperm are deposited at the anterior vagina, capacitation begins when the sperm migrates through the cervix. In species where semen is deposited in the uterus, capacitation is initiated at the uterus and completed in the isthmus of the oviduct. The process of capacitation includes activating multiple signal transduction mechanisms that lead to modification of surface molecules, alterations in the intracellular ionic concentration and activation of the enzymatic system (Fig. 23.1). The capacitation includes both membrane and cytoplasmic events. The membrane remodelling is the most pronounced event of the capac- itation that leads to increased fluidity of the phospholipid bilayer and promotes acrosomal reaction. The albumin and high-density lipoproteins of female genital tract secretions remove cholesterol from the sperm plasma membrane and alter its fluidity. The modifications in the sperm plasma membrane expose the ligands that bind with the zona pellucida. The activation of membrane-bound enzymes helps the sperm to penetrate the cumulus oophorus. The
Table 23.2 Major biochemical reactions and the role of some specific bio-molecules involved in the capacitation border=0>
| Bio-molecules | Effects | Probable mechanism |
| 1. Bicarbonate | Membrane flexibility for ion transport | Influence adenylyl cyclase (AC)/cyclic adenosine 3',5'-monophosphate (cAMP)/ protein kinase A (PKA) signalling pathway |
| 2. Calcium | Alkaline cytoplasm and phosphorylation | Activate (hyperpolarisation) membrane voltage-dependent channels and increase cAMP |
| 3. AC and cAMP | Second messenger and PKA lead to membrane remodelling, ATP synthesis | AC activate cAMP |
| 4. PKA | Hyperpolarisation of the sperm plasma membrane, intracellular alkalinisation and calcium ion trigger an acrosomal reaction (AR) | Tyrosine phosphorylation, a series of biochemical reaction, activate phospholipase D (PLD) |
| 5. PLD | Actin formation | Actin polymerisation |
| 6-8. Adenosine, fertilisation-promoting peptide (FPP), calcitonin | G proteins stimulate capacitation | Regulates AC/cAMP (initially stimulate followed by inhibiting), acts as first messengers |
| 9. Angiotensin II (AII) | Intracellular calcium ion | Indirectly regulates AC/cAMP |
| 10. Albumin | Membrane flexibility for ion transport, membrane phospholipid and lipid reorganisation | Cholesterol depletion |
| 11. Glucose | ATP synthesis | Major precursor for the glycolytic pathway |
| 12-13. Pyruvate and lactate | Glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), ATP synthesis | Regenerate cytosolic NAD+ |
| 14. Platelet-activating factor (PAF) | On capacitation by—intracellular calcium ion followed by sperm motility and on AR and embryo implantation | Signalling phospholipid acts by activating phospholipase that converts diacylglycerol (DAG) to inositol triphosphate (IP3) |
| 15. Progesterone | On capacitation and hyperactivation through PAF-mediated action, intracellular calcium, efflux of chloride and cholesterol and on AR— phospholipases and tyrosine phosphorylation of sperm proteins | Influences PAF, activates acrosome surface receptors |
| 16-18. Oestrogen, genistein and 4-tert- octylphenol (OP) | Ion channels, calcium fluxes, cyclic nucleotides (cAMP), various kinases | Acts on acrosome surface receptors for oestrogen (ESR1 and ESR2) in low concentration |
| 19-20. Seminal antioxidants (like catalase, superoxide dismutase and ergothioneine) and mitochondrial aldehyde dehydrogenase 2 | Protect sperm cell degeneration during capacitation | Reduce cytoplasmic excess concentrations of reactive oxygen species (ROS), like superoxide anions and hydrogen peroxide |
![]()
Fig. 23.1 Mechanism of capacitation and acrosomal reaction. Capaci- tation process is initiated with the increased plasma membrane fluidity and permeability for bicarbonate, depletion of cholesterol and clustering of lipid raft receptors. Albumin acts as a cholesterol acceptor. The PTK (= protein tyrosine kinase) is activated by the progesterone (PR = progesterone receptor) and ZP3 (= zona pellucida 3 proteins, ZP3R = ZP3 receptor). The activation (+) of cAMP-PKA-(protein) dependent tyrosine phosphorylation and ROS (= reactive oxygen species) production cause calcium influx within the spermatozoa followed by increased motility (hyperactivated motility) as well as the occurrence of the acrosomal reaction. (Source: Leemans et al. 2019)
alterations in the membrane protein conformation increase the permeability of the plasma membrane for calcium and bicarbonate (HCO3-) by activating T-type (voltage-sensitive) calcium channels in the sperm membrane. Increased HCO3- concentration leads to higher intracellular pH and activation of adenylyl cyclase, which in turn causes cAMP production and cAMP-dependent PKA activation. The PKA phosphorylates protein tyrosine which facilitates sperm zona binding. The γ-aminobutyric acid (GABA) acts as an inducer of both capacitator and hyperactivation in sheep, rats and humans but plays an inhibitory role in hamsters. Progesterone and oestrogen of follicular fluid act as inducers for capacita- tion in many other species like cattle, sheep, goats, pigs, horses, dogs, mice, golden hamsters and humans. These steroids facilitated the cholesterol and chloride ions efflux and increased intracellular calcium ions. Progesterone can also stimulate the capacitation process by inducing the follicular fluid’s platelet-activating factor (PAF). The capacitation is considered an oxidative process in humans, where superoxide anion (O-), nitric oxide (NO) and hydrogen peroxide (H2O2) are produced. The time requirement for capacitation to achieve sufficient spermatozoa for fertilisation is species specific (Table 23.2).
23.1.3 Hyperactivation
The hyperactivation of the spermatozoa denotes a state in which the sperm exhibit vigorous motility due to increased flagellar beating amplitude and asymmetrical beating pattern. Hyperactivation facilitates the detachment of spermatozoa from the isthmus epithelium. Hyperactivation also aids the movement of spermatozoa through the viscoelastic mucus- filled tortuous bending of the ampulla due to the flexibility of the sperm head at a wider angle. The viscoelastic substances are generally made by long-chain polyacrylamide or methylcellulose present in the tubular lumen and at the extracellular matrix of the cumulus oophorus. Hyperactivation also enables spermatozoa to penetrate the zona pellucida. The capacitated spermatozoa can bind with the zona, but only hyperactivated sperms can penetrate it. The peristaltic movement of the oviduct and the cilia-directed ductal fluid
![]()
Fig. 23.2 Molecular mechanism of hyperactivation. Schematic repre- PKA pathway. Hyper motility induces when this pathway is activated sentation of signalling pathways involved in the activation of the mam- together with the calmodulin kinase (CaMK) activation. (Image malian sperm (hyper). Progressive motility is regulated by AC/cAMP/ modified from Turner 2006)
movement also facilitate the sperm migration through the ampulla. It has also been observed that the little (about 2 ° C) warmer temperature in ampulla than isthmus favours the hyperactivation of rabbit sperm.
The hyperactivation of spermatozoa is induced by calcium either directly binds with plasma membrane phospholipids or through intracellular calcium receptor calmodulin (CaM). The Ca2+-CaM complex stimulates the activity of various enzymes like adenylyl cyclases, phosphatases, phosphodiesterases and protein kinases (PK). Activation of these enzymes, particularly PKA, causes the phosphorylation of axonemal dynein and increases ATP consumption, leading to hyper motility (Fig. 23.2).
The equine spermatozoa are unique in terms of premature natural acrosome reaction. The spermatozoa lose their acrosomal integrity immediately after the semen collection and poorly bind with zona pellucida, which hinders semen collection and preservation. The use of capacitated sperm with appropriate inducers, like progesterone, calcium ionophore and heparin, improves the sperm binding and fertilisation rate.
The two processes, capacitation and hyperactivation, make the spermatozoa completely mature and prepare them ready for fertilisation process.
23.1.3.1 Process of Sperm Release from Oviductal Epithelium
The capacitation and hyperactivation enable the sperm to detach from the oviductal epithelium near ovulation. The capacitation causes the shedding of epithelium binding proteins from the sperm plasma membrane flowed by increased flagellar movement acquired through hyperactivation. Endocrine changes that trigger ovulation have a minimal role in sperm detachment in many species. The BSP proteins have heparin-binding sites. The glycosaminoglycans released during late oestrus bind with these BSP proteins and facilitate sperm detachment. Heparin- like compounds of the oviductal fluid also bind with annexin, the oviductal receptors for BSP and cause sperm detachment. The hyperactivation of the spermatozoa is characterised by asymmetrical flagellar beating with an increased amplitude which generates sufficient force to detach spermatozoa from the oviductal epithelium.
23.1.4 Migration of Ova and Completion of Oocyte Maturation
During ovulation, the oocyte is captured by the infundibulum. This process is called ova pick-up or egg pick-up. The infundibulum surrounds the ovary, which facilitates receiving the oocyte (cumulus-oocyte complex, COC) within the oviduct. The COC moves towards the ampulla by the rhythmic movements of hair-like fimbriae and kinocilia of the fallopian tube. Kinocilia is the particular type of motile cilia abundant in the fallopian tube that assists in propelling the fluid. The time required for COC to reach the site of fertilisation is species specific (Table 23.1). The state of maturity of the oocyte is not the same in all species. In most species, the haploid secondary oocyte is ovulated in the form of an ootid with a germinal vesicle (nucleus) at its arrested meiosis II stage. In dogs and horses, the meiosis I is completed after ovulation, but the cell division continues up to meiosis II, like in other animals, before fertilisation.
About 48-96 h is required in the dog after ovulation to complete meiosis I, followed by 60-108 h for meiosis II (metaphase-II). Hence, approximately 4-7 days after LH surge, the oocyte of a dog is capable of fertilisation. Meiosis II completes when the spermatozoa interact with the ovum.
23.1.3 Process of Fertilisation
The sperm and oocyte encounter requires three critical steps, namely sperm migration through cumulus cells, sperm attachment and migration through zona pellucida, gamete fusion or karyogamy or amphimixis and block of polyspermy (Fig. 23.3). An optimum environment is required for the process of fertilisation. Different ions, namely calcium, bicarbonate, sodium and magnesium, maintain the optimum pH necessary for fertilisation.
23.1.5.1 Sperm Migration Through Cumulus Cells (If Present)
Immediately after the ovulation, the ovum is surrounded by loosely packed follicle cells called cumulus oophorus in certain species like rodents. The intercellular matrix of cumulus oophorus contains a cementing substance made of mucopolysaccharide substances hyaluronic acid. Hyaluronidase, present on the outer surface of the sperm acrosome of the
Fig. 23.3 Process of fertilisation. The events of the acrosomal reaction in the fertilisation are elaborated in steps A to E; where A = the spermatozoa come in contact with the zona pellucida of the oocyte, B = spermatozoa start a reaction with the zona pellucida, C = spermatozoa reached the perivitelline space,
D = spermatozoa (with reacted acrosome) fused with the plasma membrane of the secondary oocyte and E = nucleus of the spermatozoa enters into the secondary oocyte rupturing the vitelline membrane. The occurrence of cortical reaction is depicted with the fusion of cortical granules involving its enzymes in the perivitelline space, causing impermeable further spermatozoa after the acrosomal reaction. (Source: Monroy 2020)
![]()
Table 23.3 Egg-binding proteins (EBPs) on sperm membrane and their complement sperm receptor on zona pellucida of oocytes
| Egg-binding proteins (EBPs) | Complement sperm receptor on ZP |
| P-Galactosyltransferase | N-acetylglucosamine (GlcNAc) residues on ZP3 |
| Sperm protein-56 | ZP3 oligosaccharides |
| Zonadhesins | Zona proteins |
| Spermadhesins | Carbohydrate residues of ZP3 |
| Zona receptor kinase | ZP3 |
| Mannose-binding protein | mannose residues of ZP |
| Sperm protein-17 (Spermspecific autoantigen) | ZP3 |
| Sperm agglutination antigen-1 | Surface antigen of human sperm |
spermatozoon, helps to digest the hyaluronic acid and allows the sperm to migrate through the cumulus oophorus. The penetration of cumulus cells is of little importance in cattle as the cumulus oophorus is usually absent 3-4 h after ovulation. However, hyaluronidase is found in the bull spermatozoa. Arylsulfatase in the boar spermatozoa helps penetrate cumulus cells in this species.
23.1.5.2 Sperm Attachment and Migration Through Zona Pellucida
The oocyte’s zona pellucida consists of three glycoproteins, namely zona proteins 1, 2 and 3 (ZP1, ZP2 and ZP3). Zp1 and ZP2 are the structural proteins to maintain the integrity of zona pellucida. ZP 3 acts as the sperm binding receptor. The sperm plasma membrane also contains two zona binding sites (1) the primary zona binding region, responsible for sperm binding with zona pellucida and (2) acrosomal reactionpromoting region (ARPR), which binds with ZP3 and initiates acrosomal reactions. Several species-specific egg-binding proteins (EBPs) on the sperm membrane bind with zona pellucida (Table 23.3) of the corresponding species. Hence, the sperm of any particular species cannot fuse with the oocyte of other species.
23.1.5.2.1 Acrosomal Reaction
Acrosomal reaction is the multiple fusions between the plasma membrane and the spermatozoa’s outer acrosomal membrane to form vesicles to release the acrosome contents by exocytosis.
23.1.5.2.2 SignalTransduction
The binding of EBPs with the receptors of ZP3 activates multiple signalling cascades (Table 23.4). Two different types of receptors are present in the sperm plasma membrane that binds with ZP3. One is G protein-coupled receptor, and another is a tyrosine kinase (TK) receptor. These receptors have different second messenger systems to augment intracellular calcium levels. G protein-coupled receptor acts through cAMP and phospholipase C (PLC) second messenger system, whereas receptor tyrosine kinase is coupled with PLC.
Elevated calcium triggers the depolymerisation of the inter-membrane actin network and activation of phospholipases for exocytosis of the acrosomal contents. The acrosomal reaction begins with the multiple fusions between the sperm plasma membrane and outer acrosomal membrane that leads to the formation of many vesicles through vesiculation. The acrosomal enzymes are released through tiny pores created during vesiculation. These vesicles are sloughed after the acrosomal reaction, leaving the inner acrosomal membrane and equatorial segment intact. The secretory product of acrosome is called sperm lysine, which contains (1) hyaluronidase—helps to dissolve cumulus cells, (2) corona penetrating enzyme—it breaks the corona radiata of the cumulus-oocyte complex (COC) and (3) acrosin or zona lysine—it is a zymogen present within the acrosomal region and converted to acrosin that digests the zona pellucida. After penetration of cumulus, corona radiate and zona pellucida, a single spermatozoon fuses with the plasma membrane of the secondary oocyte. Progesterone also induces acrosomal reactions after binding with sperm surface receptors. Progesterone increases the pH of the sperm head cytosol and intracellular calcium level.
Assessment of the acrosomal integrity of sperm is one of the essential evaluation criteria for semen analysis and can do immediately after semen collection. The hypoosmotic swelling (HOS) test is routinely used to assess acrosomal integrity. Monoclonal antibodies and indirect
Table 23.4 Signal transductions to initiate the acrosomal reaction
| Receptor | Second messenger system | Signal transduction |
| G protein-coupled receptor | cAMP | Activation of adenylyl cyclase (AC) leads to cAMP production and protein kinase A (PKA) activation. The PKA activates a voltage-gated Ca2+ channel at the outer acrosomal membrane to allow the entry of Ca2+ from the acrosome to the cytosol. |
| G protein-coupled receptor | Phospholipase C (PLC) | PLC hydrolyses phosphatidyl-inositol bisphosphate (PIP2) to diacylglycerol (DAG) and inositoltrisphosphate (IP3). DAG helps in the translocation of protein kinase C (PKC) at the plasma membrane. PKC stimulates voltage-gated Ca2+ channel (L) in the plasma membrane, leading to more intracellular calcium. |
| Receptor tyrosine kinase | Phospholipase C (PLC) |
Table 23.5 Sperm proteins and their receptors on oocyte surface to facilitate gamete fusion
| Sperm proteins | Receptors on oocyte surface |
| Fertilin α (ADAM1) | CD9 |
| Fertilin β (ADAM2) | α6β1 integrin, α9β1 integrin |
| Cyritestin (ADAM2) | CD9 |
| CD46 | β1 integrin |
| Izumo | Juno |
immunolabeling techniques are also used to evaluate the same.
23.1.5.3 GameteFusion
Once the sperm has traversed through zona pellucida, the head moves into vitelline space to interact with the vitelline membrane. The penetration of the vitelline membrane activates the ovum, and the resumption of meiosis occurs. The female pronucleus is formed after the completion of meiosis. The equatorial region of the sperm is incorporated into the plasma membrane of the ovum. Several molecules have been identified in sperm and ovum that facilitate gamete fusion (Table 23.5).
Other molecules responsible for gamete fusion are spermosin, HYAL5T, angiotensin-converting enzyme 3 (ACE3), trypsin-like acrosin and SPAM1. The beating of sperm tails stops immediately after the sperm-egg fusion. The fusion results in actin polymerisation and the extension of microvilli. The cytoplasm of the oocyte starts swelling and forms a fertilisation cone. Then the sperm is drawn by the microvilli of the egg, and the sperm nucleus, together with other organelles, is incorporated into the cytoplasm of the oocyte. All the cellular organelle of the spermatozoon, except periacrosomal materials, are engulfed by the fertilisation cone. The periacrosomal materials are infused into the oocyte cytoplasm, which favours oocyte activation for resumption of meiosis II. The nucleus and acrosomal tubules containing centrioles and mitochondria of the mid-piece of spermatozoon remain in the cone. Only the nucleus and centrioles are involved in the fertilisation process, and other structures do not have any role. The acrosomal tubule dissolves, and the centriole divides into two halves which form the mitotic spindle. The mammalian oocyte does not have any centriole. The nuclear envelope of sperm is disintegrated, followed by chromatin decondensation.
The factor responsible for chromatin decondensation is called the male pronucleus growth factor. A new nuclear envelope of sperm develops within the oocyte cytoplasm, forming a male pronucleus. The male and female pronuclei migrate to the centre of the ovum. Then nuclear membranes of both male and female pronuclei disperse, and chromosomal intermixing occurs. This process is called karyogamy or amphimixis. It occurs within 24 h of ovulation in cattle. A diploid zygote is formed due to the karyogamy process. The formation of zygotes denotes the end of fertilisation. The chromosomes aggregate in the prophase of first cleavage division, leading to zygote formation and the restoration of the diploid state. The fusion of male and female gamete is called syngamy.
In polytocous animals, more than one oocyte is ovulated and simultaneously fertilised. Sometimes, two embryos can develop in monotocous animals due to specific fertilisation abnormalities (discussed in detail in the fertility-related abnormalities section). In the case of polyspermy (more than one sperm involved in fertilisation), four cells are developed with improper numbers and types of chromosomes. These cells may die or undergo abnormal development.
23.1.5.4 Block to PolyspermyZEgg Cortical Reaction
Polyspermy is the process of fertilisation of an oocyte by more than one spermatozoon. Polyspermy usually leads to embryo death. Immediately after the entries of spermatozoa through the zona pellucida, the surface of the ovum changes continuously to prevent further sperm binding, called block to polyspermy. The process is mediated by egg cortical reaction, where the exocytosis of the cortical granules (CG) present below the oolemma causes zona block. The block of polyspermy usually occurs at the zona pellucida in most species. But, in rabbits, a secondary block at the vitelline membrane also occurs. The block of polyspermy occurs in two steps fast and a slow block.
23.1.5.4.1 FastBlock
Within a second after the fertilisation, the membrane potential of the oolemma changes to the depolarised state by a massive influx of Na+ ions. It is called the fast block to polyspermy as the sperm cannot penetrate a membrane where the potential is more than -70 mV. The fast block of polyspermy is intended to prevent sperm attachment to the oocytes. The oolemma undergoes rapid repolarisation within a minute through K+ leakage.
23.1.5.4.2 Slow Block/Cortical Reaction
In this process, the cortical granules release their content to modify the extracellular matrix of the zona pellucida. It acts as a permanent barrier to prevent further entry of spermatozoa. In cortical reaction, secretory vesicles are fused with the oolemma and release their contents (Fig. 23.3). Two classes of proteins, namely soluble NSF-attachment protein receptors (SNAREs), are involved in the translocation of cortical granules and membrane fusion. SNAREs have two components, vesicular (v) and target membrane (t), found in cortical granules and oolemma. Vesicle-associated membrane protein (VAMP) and
Table 23.6 Cortical granular contents and their role in block to polyspermy
| Cortical granule contents | Role |
| Proteinases Tissue-type plasminogen activator (tPA) | Zona hardening and blocks sperm penetration by converting plasminogen to plasmin |
| ZP2 proteinase | ZP2 (120 kDa) is converted to ZP2f (90 kDa) by proteolysis. ZP2f causes zona hardening. |
| Ovoperoxidase | Catalyses the tyrosines cross-linking in the zona resulting zona pellucida hardening |
| Calreticulin | Blocks the carbohydrate moieties of glycoproteins required for sperm-oocyte interaction |
| N-Acetylglucosaminidase | Cleaves the terminal N-acetylglucosamine residues of zona protein to prevent sperm binding |
| p32 | Prevents sperm binding |
| Peptidyl arginine deiminase (PAD/ABL2antigen/ p75) | Forms an extracellular matrix in the perivitelline space called cortical granule envelope |
| Glycosaminoglycans | Attracts water into the perivitelline space and allows it to expand and form the hyaline layer |
Synaptotagmin are v-SNAREs situated at the vesicle membranes. Syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) is t-SNARE found in oolemma. The interaction between v and t SNAREs results in membrane fusion. The cortical granules contain several proteins and enzymes that alter the zona pellucida and vitelline membrane to facilitate block to polyspermy (Table 23.6).
23.1.5.4.3 Molecular Mechanism of Egg Cortical Reaction
Cortical granules’ exocytosis involves a calcium-dependent pathway. The binding of sperm with ZP3 initiates G protein- coupled receptor signalling and activation of PLC, which cleaves phosphatidylinositol 4,5-biphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). PIP2 and DAG act as second messengers. IP3 binds to its endoplasmic reticulum receptors and facilitates the release of Ca2+ from the endoplasmic reticulum. The Ca2+ binds its receptors at the endoplasmic reticulum around the cortical granules and results in more Ca2+ releases (Ca2+-induced Ca2+ release, CICR process), and a wave-like calcium spreading occurs to rupture the granules. DAG induces PKC activation, which phosphorylates ion-exchange proteins for Na+ to H+ and increases Na+ and H+ output. The pH of the ovum rises from 6.8 to 7.3, favouring the awakening of oocytes from metabolic inertia. The CG exocytosis process involves two important regulatory proteins, calmodulin (calcium-binding) and gelsolin (involved in restructuring cortical F-actin). Due to insufficient CG, polyspermy occurs in particular mammals, like pigs and marsupials. IP3-mediated granular exocytosis is also absent in these species.
23.1.5.5 Activation of Zygote and Initiation of Mitosis
The zygote becomes activated immediately after fertilisation. A zygote is totipotent and can form all cells, including an extraembryonic membrane. The fertilised ovum undergoes a series of events, like degradation of maternal products (RNA and protein), post-translational regulation and epigenetic reprogramming. All these events are collectively called maternal-to-zygotic transition. Ca2+ plays a central role in activating the oocyte from meiotic arrest and triggers the embryonic development programme. High intracellular Ca2+ inside the oocyte coincides with the fertilisation through Ca2+-induced Ca2+ release (CICR process). Mature oocytes are in M II block due to the action of the M-phase-promoting factor (MPF) that forms a complex with cyclin B and cyclin- dependent kinase p34cdc2. High MPF activity leads to stabilisation of the meiotic spindle and chromatin condensation. The rise of Ca2+ leads to proteolysis of cyclin B and inactivation of MPF to resume meiosis. The amplitude and duration of Ca2+ spikes required for oocyte activation are species specific. In mice, Ca2+ spike occurs at an interval of 10 min. In humans, pigs and cows, Ca2+ spike occurs every 30-60 min.
23.1.5.5.1 Zygotic Genome Activation (ZGA)
The initiation of gene expression after fertilisation is called zygotic genome activation (ZGA). Before the activation of the oocytes, the transcription arrests as the transcription factors are unable to bind with their motifs due to chromatin condensation. Several pioneer factors facilitate the binding of transcription factors with their motifs to induce ZGA. The first identified essential pioneer factor in influencing ZGA is Zelda. It generates the transcription of hundreds of genes by histone acetylation and nucleosome remodelling that facilitates the binding of other transcription factors. Other pioneer factors involved in ZGA include Nanog, Oct4 and SoxB1. Nanog and SoxB1 are involved in nucleosome destabilisation, and Oct4 is the key factor in inducing pluripotency (the ability of the individual cell to form all tissue lineages).
23.1.4 Fertility-Associated Proteins
Several species-specific proteins are involved in different aspects of acrosomal reaction and gamete fusion. They are called fertility associated proteins and are used as a biomarker to assess fertility in animals. The sperm acrosome-associated 1 (SPACA1), a tyrosine-phosphorylated protein, is required for acrosomal reaction in bull, boar and mice. Another acrosomal protein, Izumo sperm-egg fusion 1 (IZUMO1), involves an acrosomal reaction and gamete fusion in bull and mouse. The sperm nuclear protein protamine 1 in bull and protamine 2 in primates and rodents are essential in protein synthesis during early embryogenesis. Some sperm transmembrane proteins like the adenylyl cyclase 10 (ADCY10, a bicarbonate sensor), osteopontin (Ca ion-binder) and Na+/K+-ATPase found in bull and humans involves in acrosomal reaction for ionic exchange.
23.1.5 FailureofFertilisation
Improper ovulation, obstruction in the oviduct, abnormal oocyte and ovarian adhesions are the major inter factors of fertilisation failure in animals. In assisted reproductive technology, inappropriate prediction of ovulation time and insemination techniques are the major causes of fertilisation failure. Ovulation-related disorders include delayed ovulation, silent heat, anovulatory oestrus, poor managemental practices, malnutrition and environmental factors, like heat stress. Pathological conditions like ovarian cyst and endometritis may also lead to fertilisation failure. The animal can be considered a repeat breeder when it fails to conceive after repeated insemination attempts despite its normal reproductive cycle. Dietary supplementation of omega-6-rich polyunsaturated fatty acids (PUFAs) as calcium salt directly affects the oocyte to increase male sex offspring in cattle.
23.1.7.1 Development of Twin
The development of twins in monotocous species is considered abnormalities. When two offspring are born at the exact birth, they are called twins. There may be three types of twin, viz. identical twin, fraternal twin and semi-identical twin.
23.1.7.1.1 IdenticalTwin
The identical twins are also called monozygotic twins, where a single oocyte is fertilised by a single spermatozoon leading to the formation of a single zygote. The single zygote develops up to blastocyst, but the inner cell mass splits into two parts to develop two separate foetuses. Embryo-splitting techniques in in vitro fertilisation intends to develop such type of twins. The monozygotic twins are phenotypically identical, and hence, they are called identical twins.
23.1.7.1.2 FraternalTwin
In fraternal twins, two separate oocytes fertilise independently, followed by the formation of two zygotes. They undergo a separate implantation process in the uterus. Hence, it is called a dizygotic twin. The term fraternal twin describes two different spermatozoa used in fertilisation. Phenotypically they may or may not be identical but may have diverse sequences on each chromosome and other sex; hence, it is called non-identical twins. Such incidence is due to hyperovulation, often seen in advanced age. Genetics and nutrition may be predisposing factors for fraternal twin development.
23.1.7.1.3 Semi-identical Twin
The occurrence of semi-identical twins is rare. A semiidentical twin develops when an unfertilised oocyte is mitoti- cally divided into two oocytes and fertilised separately by different spermatozoa. The offspring may be of another sex with non-identical genetic and phenotypic characteristics; hence, it is termed semi-identical twins and occurs in various mammals like cattle, sheep, dolphins, elephants and humans.
23.2
