FERTILIZATION

The union of a spermatozoon and ovum marks the beginning of a new life.

Capacitation. The chemical basis of capacitation is uncertain. When sperm make their way through the epididymis, and when mixed with accessory secretions, decapacitation factors (eg., caltrin, a calcium-transport inhibitor) are adsorbed onto the plasma membrane covering the acrosome; in this state the acrosome is inhibited from liberating its fertilizing enzymes too early. When spermatozoa are bathed in secretions of the female genital tract (or washed in an appropriate media) they become capacitated and capable of penetrating the outer investments of the ovum. Ova secrete factors that attract capacitated sperm.

Acrosomal reaction and fusion. The corona radiata persists around ovulated ova of most mammals. Spermatozoa must pervade the corona radiata and the zona pellucida before reaching the ovum proper; they do so by releasing hydrolytic enzymes from the acrosome - the corona-penetrating enzyme (hyaluronidase) and acrosin (a trypsin-like protease which digests the zona pellucida). The acrosomal reaction is initiated when spermatozoa come in contact with the corona radiata or zona pellucida. The acrosome is decomposed during sperm passage through the zona pellucida.

In monotremes, marsupials, sheep, and cattle, coronal cells become almost completely dispersed from the ovum shortly before or soon after ovulation (Figure 5-15). The zona pellucida is the only barrier through which spermatozoa must pass in these species.

Definitive binding of sperm cells to the zona pellucida involves specific interactions between receptors in the sperm plasma membrane and exposed zona adhesion molecules (eg., integrins). Initial contact and fusion of a spermatozoon with the secondary oocyte occurs near the equatorial border of the head of the sperm cell (Figure 5-16). The plasma membrane of the head and tail of the spermatozoon is incorporated into the vitelline membrane of the ootid. The nuclear membrane of the spermatozoon dissipates and its chromatin is released into the ooplasm; this material becomes surrounded with a new membrane to form the male pronucleus. Nuclear membranes of male and female pronuclei soon disintegrate and their chromosomes intermingle (syngamy) before initiation of the first mitotic division of the newly formed zygote.

Hypermotility of spermatozoa provides a physical force necessary for incremental movements through the enzymatically disrupted obstacles of the ovum. In humans and some other species there is a slight excess in male births; this may stem from an enhanced fertilization rate by more motile Y-bearing spermatozoa.

Zona block. An ovum can be assailed by numerous spermatozoa. The ovum plays an active role in preventing fertilization by more than one sperm cell (ie., polyspermy). A wave of intracellular calcium release is induced upon fusion of a spermatozoon with the vitellus. The ovum responds by secreting enzymes and an acrosin inhibitor from cortical granules into the perivitelline space (Figure 5-17). Enzymes change the physicochemical properties of the zona pellucida so that additional spermatozoa are halted in their quest toward the vitellus. The (depolarized) vitelline membrane itself becomes refractory to sperm attachment.

In vitro fertilization. Assisted techniques of reproduction, such as IVF-ET, have become almost routine in seedstock animal breeding and research applications and in specialized human clinics.

With livestock (eg., cattle), donor females are superovulated to increase the number of ova/embryos collected to a single recovery. Hormonal preparations are used that stimulate growth of multiple follicles (PMSG or FSH). Problems with superovulation are that responses can be widely variable and some oocytes recovered under these conditions are abnormal (ovulation from a follicle that would have otherwise undergone atresia?); the latter situation can lead to genetic defects in the offspring that are not currently capable of being screened during microscopic inspection. Recipient animals should be synchronized to estrus with the donor so they are in the same stage of the estrous cycle at the time of transfer of (fresh) embryos (Figure 5-18).

Surgical and nonsurgical methods of collection have been developed. Nonsurgical procedures are preferred. Embryos are collected from cattle and horses with a Foley catheter (Figure 5-19). One can expect to recover about 50% of embryos resulting from a superovulation (eg., about five of ten in the cow). Ova for IVF are either aspirated directly from preovulatory follicles or flushed from the oviduct. Chimeric embryos can be created by IVF using gametes of closely related species (eg., a sheep-goat mosaic).

Embryos to be transferred fresh can be maintained in sterile media at 37C for up to 12 hours. Best results are obtained when embryos are transferred within two hours of recovery. Embryos remain viable for up to two days at reduced temperatures (10C). Freezing and storage of embryos is performed, but a 50% reduction in viability upon thawing should be anticipated.

Transfer of embryos also can be accomplished surgically or nonsurgically. An embryo is usually placed into the uterine lumen using methods described for AI (eg., the bovine embryo is placed in a straw and injected using an inseminating gun). It is recommended that the embryo be introduced into the uterine horn ipsilateral to the CL.

Common applications of IVF-ET in human medicine include tubal blockage (Figure 5-20) and unexplained infertility; a representative protocol for this procedure is outlined in Figure 5-21. Three to five oocytes are typically collected. One or two (to optimize chances of a successful implantation in women over 35) fertilized eggs (two- to eight-cell stage) are usually transferred. Embryos exhibiting morphological signs of abnormality (eg., fragmentation or polyspermy) are discarded. Viable embryos that are not transferred can be frozen and stored for future use. Zona drilling or ICSI are used in severe cases of male infertility (when routine IVF fails).

A hazard of ovarian hyperstimulation in human IVF (and other ovulation induction) programs is postovulatory ovarian enlargement (ie., multiple cystic and hemorrhagic follicles and CL) and acute vascular leakage. Moderate to severe complications (usually < 10%) can be manifested by ascites, hydrothorax, hydropericardium, pulmonary edema, and hypovolemia. Treatment includes bed rest and stabilizing third-space fluid shifts. Surgery (ovarian wedge resection or ovariectomy) is reserved for cases of severe ovarian torsion and intraperitoneal hemorrhage.