Fertilization and Implantation

Sperm must travel a considerable distance in comparison to their size from the site of production to the site of fertilization, passing through a range of environments along the way. The male gonads are the site of sperm production and prepare the seminal plasma for ejaculation while the female receives the semen during copulation and provides the egg and site of fertilization. Under hormonal control spermatozoa move from the testes to the epididymis, then deferent duct, and are finally excreted through the urethra of the male (Dyce, Sack, & Wensing, 2002). In the female, they must navigate the vagina, cervix, and uterus in order to reach the uterine tubes for fertilization.

Sperm production, or spermatogenesis, is regulated by the pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. GnRH acts on the anterior pituitary to stimulate the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). FSH and LH work in tandem to stimulate and sustain sperm production throughout the lifetime of a male. Spermatogenesis begins in the seminiferous tubules that make up the testicles. LH stimulates interstitial Leydig cells to produce testosterone which is then converted to dihydrotestosterone and estrogen in the sertoli cells under the action of FSH. The conversion of testosterone is directly facilitated by androgen binding protein (ABP), which is produced in response to FSH interaction with receptors on the surface of the sertoli cells (Hafez, 1993). FSH is subsequently responsible for the completion of sperm release into the seminiferous lumen and secretion of inhibin. Inhibin has a negative feedback effect on FSH but not LH. So LH continues stimulating testosterone production until the concentration of testosterone is high enough to inhibit the release of GnRH, LH and FSH by negative feedback on the pituitary and hypothalamus (Hafez, 1993). Spermatocytes develop as they migrate through the sertoli cell, the head and tail becoming progressively more distinctive.

The spermatozoa released into the lumen of the seminiferous tubules are immotile and immature. The epididymis is an elongated organ running longitudinally along the testes and is the site of sperm maturation. Spermatozoa are moved from the seminiferous tubules to the epididymis by a combination of muscular contraction - by the myoid cells and testicular capsule - and action by the cilia lining of the efferent ducts (Hafez, 1993). Pressure flow is also created by secretions from the sertoli cells and rete testis and the resorption of fluid by the lining of the efferent ducts (Dyce, Sack, & Wensing, 2002). Secretions from the sertoli cells makes up the majority of the testicular fluid, and ABP is also contained in this fluid. It forms a complex with the androgens produced by the leydig cells and may assist in transit of the androgens into the epididymis. Spermatozoa mature as they move up the epididymis until they reach the caudal (tail) portion where they are stored. Peristalsis of the vas deferens gradually moves sperm to the ampullary region. Gradual seeping of sperm into the urethra occurs in sexually inactive males, leading to potentially high levels of spermatozoa in the urine (Dyce, Sack, & Wensing, 2002). The much faster form of sperm ejection occurs during copulation in the sexually active male. Ejaculation is a reflex expulsion of spermatozoa and seminal plasma from the male reproductive tract. It is under sensory control, receiving stimulation from nerves in the glans penis which generate neural impulses during excitement by copulatory action (Husveth, 2011). The major muscles involved in ejaculation are the urethralis and bulbospongiosus. Their contractions, stimulated by the nervous system, move the semen through the urethra, generating a forceful ejaculation into the vagina or cervix of the female (Dyce, Sack, & Wensing, 2002).

Seminal plasma serves as carrier and protector of spermatozoa in natural mating, especially in the ewe and cow where sperm are deposited in the vagina. Source of seminal plasma constituents varies with species. Prostate, vesicular and bulbourethral glands pour their secretions into urethra where, at the time of ejaculation, they are mixed with fluid suspension of sperm and ampullary secretions from the ductus deferens. The only accessory gland common to all mammals is prostate (Hafez, 1993). The seminal plasma contains a mixture of compounds including antibodies and glucose, presumably to provide the sperm with a source of energy and also protect them and the female from infection by microbes. Once the semen enters the vagina, sperm concentration is depleted at a number of stages so that only a small fraction of the original sperm arrives at the site of fertilization.

The vagina is the most common site of sperm deposition. Here, thick mucosal secretions trap or slow the sperm, and sperm capacitation may begin here. Capacitation is the process of changes being made to the components of the outer acrosome and plasma membranes so that acrosomal enzymes can later be released and activated within the female (Husveth, 2011). When sperm is first ejaculated its ability to fertilize is poor. Capacitation involves an influx of extracellular calcium, increase in cyclic AMP, and decrease in intracellular pH. Some of these changes are induced by the environment of the female reproductive tract (Bowen, 2000). When estradiol levels are high (animal in heat/breeding), squamous cells in the vagina mature and become cornified, causing epithelial thickening. During the luteal phase, number of precornified cells increases, number of leukocytes and amount of cellular debris increase as mature squamous cells are shed (Brzyski & Knudtson, 2013). Leukocytes engulf the sperm and digest them, so sperm viability is likely to remain higher if breeding occurs in the late follicular phase where leukocyte numbers are still low and fewer cells have become cornified. Mucus production is also heightened in the luteal phase, further inhibiting movement of spermatozoa in the female reproductive tract. Mucus provides a suitable environment for the maintenance of metabolic activity of spermatozoa, helping to maintain the integrity of the gamete. Transport of spermatozoa into the uterus may influence capacitation because sperm are separated from an excess of “decapacitation factor” and from other enzyme inhibitors in the seminal plasma (Hafez, 1993).

To enter the uterus, the sperm must pass through the cervix. The cervix acts as a barrier that limits access to uterine cavity. It has many folds that filter out immotile sperm, reducing the sperm count by huge proportions. High estradiol concentration – being produced by dominant follicle in late follicular phase - increases cervical vascularity, elasticity, and salt concentration. As a result, high fertility is observed during the follicular phase. High elasticity of the mucus allows for ready transport of sperm as compared to the luteal phase. Progesterone makes mucus thicker and less elastic, decreasing success of sperm transport during the luteal phase and therefore reducing fertility (Brzyski & Knudtson, 2013).

It takes 2.5 minutes to reach site of fertilization in cattle, which would take 1.5 hours to ‘swim’ the distance (Husveth, 2011). Other factors must be in play to allow such rapid transport of the seminal fluid from the vagina to the oviduct. Increased oestrogen levels during the follicular phase upregulates oxytocin receptors both at the uterus and uterotubal junction (Gimpl & Fahrenholz, 2001). The resultant oxytocin uptake leads to peristalsis of subendometrial layer of muscular wall in early and mid follicular phases (Kunz, Noe, Herbertz, & Leyendecker, 1998). Rhythmic peristaltic contraction of the non-pregnant uterus, reaches a maximum just prior to ovulation, controlled by oestradiol secretion from dominant follicle (Kunz, Beil, Huppert, & Leyendecker, 2007). The result is rapid sperm transport from the vagina to the Fallopian tubes. Within the uterus, sperm is separated from seminal plasma and transported to oviduct (Hafez, 1993).

During the follicular and luteal phases of the estrous cycle, uptake of particles into the uterus is observed. During the follicular phase, only transport into the oviducts is observed. Stimulated by the uptake of oxytocin, the predominant transport is into the tube ipsilateral to the ovary containing the dominant follicle (Zervomanolakis, et al., 2007). Ciliated cells facilitate movement of gametes (and embryo after fertilization) within the oviducts. Progesterone causes decrease in cilia beating frequency, and modulates transport speed of the cumulus complex in order to increase likelihood for fertilization to take place within the ampullary region (Bylander, Lind, Goksor, Billig, & Larsson, 2013). Oestradiol acts on the uterotubal junction, causing it to close with a sphincter-like action prior to ovulation. Sperm numbers are reduced further at this barrier. The ampullary-isthmic junction is a close control point of egg transport in the oviduct. It is also the site where the sperm plasma membrane changes (acrosome reaction), completing sperm capacitation. Sperm motility in oviductal fluid increases thereafter, so at the ampulla sperm are equipped to penetrate the corona radioata and zona pellucida when contact is made with the egg. It is in the ampulla at fertilization that acrosomal proteinases are released from the sperm head.

Overall, most spermatozoa are lost at the cervix, uterotubal junction, and oviductal isthmus (Hafez, 1993). Any spermatozoa that reach the fimbrae may be released into the peritoneal cavity and lost. After coitus, the intraluminal leukocyte concentration increases dramatically and phagocytize excess spermatozoa. Many sperm are eliminated by retrograde flow out of the vagina, but a fair portion remain trapped in the uterus and must be taken up into phagocytic vacuoles and digested by leukocytes (Hafez, 1993).

Sperm Transport in Male and Female Reproductive Tracts

Ovum development and Embryo Implantation

A female is born with a finite number of egg precursors, or germ cells. They develop in the ovaries of the fetus during gestation. Through the fourth month of gestation, primordial oogonia proliferate by mitosis, although at three months some begin to undergo meiosis so that they are haploid instead of diploid (contain half the number of chromatids of a diploid cell). At seven months into gestation, all viable germ cells develop a surrounding layer of granulosa cells, forming a primordial follicle. At this stage, the oogonia are arrested in meiotic prophase and are referred to as primary oocytes (Brzyski & Knudtson, 2013). Follicular growth is stimulated by FSH, controlled by the pulsatile release of GnRH from the hypothalamus.

The onset of puberty is marked by a surge in the release of GnRH, which triggers a waterfall of downstream events. Mullerian Inhibiting Substance (MIS) is produced in the female at the same levels found in adult males only at puberty. MIS inhibits and blocks meiosis and spontaneous breakdown of the germinal vesicle ovarian oocytes (Hafez, 1993). This means that primary oocytes are more stable than during early development and can progress into a dominant follicle. Estrous cycles in the female begin at puberty. During the estrous cycle, anywhere from three to thirty primary oocytes are recruited for accelerated growth in the ovary stimulated by FSH. Follicles produce oestradiol, which promotes LH and FSH production but inhibits their release so that there is a negative feedback effect on follicle production. Granulosa cells surrounding the oocyte produce inhibin, which inhibits FSH release from pituitary without altering LH release (Hafez, 1993). Inhibin is partially responsible for atresia of the non-dominant follicles. The result is a series of follicular waves, at the end of which a dominant follicle develops, releases an oocyte at ovulation, and promotes atresia of other recruited follicles. Oestradiol is produced by the dominant follicle at a peak just prior to ovulation. The oestradiol peak has a positive feedback effect on the preovulatory center of the hypothalamus, stimulating the release of GnRH. LH secretion ensues and in turn stimulates enzymes in the ovary that initiate breakdown of the dominant follicular wall to release the mature ovum within 16 to 32 hours. The LH surge also triggers completion of the first meiotic division of oocyte within about 36 hrs.

A corpus luteum forms on the ovary in place of the ruptured follicle, which lasts about 14 days and degenerates under the action of prostaglandin if no pregnancy occurs. It produces progesterone in increasing quantities, peaking 6-8 days after ovulation. Progesterone stimulates development of secretory endometrium, which is needed for embryonic implantation. Because levels of estradiol, progesterone, and inhibin are high during most of luteal phase, LH and FSH levels decrease and further follicular growth is inhibited. If implantation occurs, the corpus luteum does not degenerate but functions in early pregnancy, supported by chorionic gonadotropin that is produced by developing embryo (Hafez, 1993).

In order to reach the oviducts from the ovary, the newly released oocyte must be caught by the fimbria and travel down the infundibulum to reach the ampulla. At ovulation, the action of progesterone and oestrogen cause the fimbriae to engorge with blood, become distended, and the ends become translucent. They are brought into close contact with the ovary by muscular action of mesotubarium, creating a net. The mass of cumulus oophorus erupting from the ovary contain the oocyte. Corona cells adhere to the ovarian stigma and remain attached until removed by action of kinocilia of the fimbriae (Fribourg, Lausanne and Bern Universities, 2016). The ovary and oviduct are held in position by ligamentum ovarii proprium (ovarian ligament) and mesovarium (Hafez, 1993). Movement of cilia in the uterine tube generates a current that causes catching of the ovum, helped down uterine tube by contractions of the fimbriae and utero-ovarian ligaments. Cilia motility plays a major role of movement of the ovum. Contractions are partly controlled by estrogen/progesterone ratio, but are also under neurohormonal mechanistic control, so contractile activity of fimbriae is stimulated at time of copulation (Hafez, 1993). The fertilized eggs must reach uterus at an appropriate progestational stage of the estrous cycle, so rate of egg transport is closely monitored. Transport from the infundibulum to ampullary-isthmic junction is faster than the rate of transport through isthmic portion (Fribourg, Lausanne and Bern Universities, 2016). Suspension of the egg in the ampullary region allows fertilization to take place, and this arrest is facilitated by sphincter-like function of ampullary-isthmic junction. Oestrogen is responsible for the primary endocrine control of the tightening of the junction. Smooth muscle contraction is regulated by myogenic pacemakers, coupled in time and space so they do not force ova transport to the uterus prematurely. The egg or fertilized zygote is moved to the uterus by a combination of tubal peristalsis, ciliary activity, and fluid currents and countercurrents within the lumen. As it moves down the oviduct toward the uterus, the fertilized embryo undergoes mitosis, so that it becomes a morula of cells, and then a blastocyst forms. Prostaglandins affect smooth musculature of oviducts and uterus, the formation and release of which is controlled by nerve activity. The sympathetic nervous system then facilitates a drop in oviductal motility around 72 hours post-coitum, by which point the ova of most species are in the uterus. By 80 hours after ovulation, the blastocyst reaches the uterine cavity and implants in the endometrium 3-7 days after fertilization (Hafez, 1993) .

Implantation of the blastocyst is dependent on several factors being present in the uterine environment. PDGF (platelet derived growth factor), a glycoprotein, supports the growth of serum-dependent cells. It is secreted by some blastocysts and is present in uterine secretions, facilitating successful implantation. (Hafez, 1993) It is interesting to note that the uterine environment is not necessary to maintain a pregnancy. The fetus is capable of surviving by placental fusion with peritoneal cavity, though it is rare.

Successful Fertilization depends on Timing of Estrogen and LH peaks

When a paternal gamete (sperm) fuses with the maternal gamete (egg), fertilization has successfully occurred. The limiting factor is the length of life of the ovum once the animal is on heat. It has roughly 12-24 hours of viability in comparison to the fairly rigorous sperm which may be viable for up to a few weeks. The unfertilized egg rapidly loses viability by the time it reaches isthmus and is completely non-fertilizable after reaching the uterus (Hafez, 1993). Fertilization of aged eggs may lead to abnormalities in the offspring, or a high percentage of nonviable embryos. Guinea pigs were bred with increasing egg age, resulting in a high percentage of abnormal pregnancies and decrease in litter size as age of egg increases prior to fertilization. Fertilization of aged eggs in swine is also associated with polyspermy and thus abnormal embryonic development. Aging of the egg may cause abortion, embryonic resorption, or abnormal development of the embryo (Hafez, 1993). Such consequences would obviously cause great economic loss to the farmer. Correct timing means of LH and oestrogen peaks, as well as time of insemination, means sperm are present in the female reproductive tract when the egg is released.

The uterus needs priming for heat and ovulation. Estrogen causes both hypertrophy and hyperplasia of cells in the endometrium and myometrium (Hafez, 1993). Estradiol levels increase as follicles develop, causing the basal uterine layer to regenerate the spongiosa and epithelial layers of the endometrium to its maximum thickness late in ovarian follicular phase. Estradiol also causes thickening of mucosa, and glands lengthen and coil. By the time ovulation occurs, the endometrium will be in the early secretory phase. Mucus will still be elastic and ideal for transporting sperm quickly to the ampulla of the oviduct for fertilization as well as being able to maintain a stable environment for embryo implantation (Brzyski & Knudtson, 2013). Estrogen has negative feedback effect on tonic center of hypothalamus, and positive effect on pre-ovulatory center (Hafez, 1993). This means that as follicles develop and produce oestradiol, they are inhibiting the release of GnRH under the control of the tonic center of the hypothalamus, so FSH and LH secretions are inhibited. Follicles are produced in waves, even during one cycle, until a single follicle dominates.

As the estrous cycle reaches the late follicular phase, the dominant follicle produces a surge of oestradiol which performs a number of functions. The most obvious function is to bring the animal on heat by acting on the central nervous system to induce behavioural estrus (Hafez, 1993). It is essential that the female shows heat before ovulation so that the male has a chance to mate with her and provide a sperm presence in her reproductive tract before the egg travels to the uterus.

The other major function that the oestradiol surge serves is to have a positive feedback effect and cause the release of GnRH from the hypothalamus so that LH is released in large quantities. LH acts on the dominant follicle, stimulating the action of enzymes to break down the follicular wall and release the oocyte. LH then facilitates luteinization of the corpus luteum, which begins producing progesterone in increasing amounts. Progesterone (from CL during luteal phase) stimulates the endometrial glands to dilate, fill with glycogen, and become secretory. At this stage the egg has begun its journey down the oviduct. Decreasing p4 and E2 cause stroma to become edematous late in luteal/secretory phase, blood vessels necros, causing bleeding and menstrual flow (if no implantation occurs)(Brzyski & Knudtson, 2013).

Estrogen increases amplitude and frequency of contraction by increasing affinity of uterus to oxytocin and PGF2a. Smooth muscle contractions of the endometrium is stimulated while the frequency of the beating of the cilia decreases significantly compared to its previous motility. Delayed cilial beating is important because the egg must not travel too quickly. Slower transport is ideal for fertilization, and allows time for enzymatic processes induced by the sperm during fertilization in the ampulla of the fallopian tubes to mature before implantation.

Improper timing of the hormonal stimulus could lead to a number of deformities in the embryo, as mentioned before. The discussion also demonstrates that oestradiol plays a crucial role in speed of egg and sperm transport, and if those two do not line up, the sperm may not have access to the egg while it is still viable. Another possibility of poor timing may be that the infundibulum is not fully extended when ovulation occurs. The egg may be lost in the peritoneal cavity, and will usually degenerate and be absorbed. But in rare cases, it may result in ectopic pregnancy (Hafez, 1993).