The Ins and Outs of Sperm Management. Part I - Evaluation of Sperm Function

Introduction  

The uninformed might describe a spermatozoon as a specialized, yet simple, cell with only one role to fulfill… that of fertilization. While fertilization is the endpoint of spermatozoal function, this cell must be extremely sophisticated and adaptable to achieve this task, and the process involves a series of highly coordinated cellular and molecular events. Following is a list of some requirements ascribed to a  mammalian spermatozoon:  

1. Loss of most organelles and cytoplasm during formation the testis and maturation in the epididymis (requires a host of intracellular and intercellular signaling events)  
2. Remodeling of spermatozoal chromatin within the epididymis as a protective mechanism against  environmental injury (requires repackaging of nuclear DNA into a highly condensed form through the aid of specialized proteins termed protamines)  
3. Plasma membrane alterations within the epididymis to yield proteins important to fertilization (requires various enzymatic-linked alterations of existing proteins, as well as uptake of proteins from epididymal fluid or from the epididymal epithelium)  
4. Passage through the uterus and uterotubal junction of the female at the time of insemination (requires activated flagellar movements and protection against immunologic attack) 
5. Binding to oviductal epithelial cells to form a spermatozoal reservoir (requires specific cell-cell attachment, possibly mediated through spermatozoal surface carbohydrate-binding proteins,  termed lectins).  
6. Acquisition of additional maturational changes, collectively termed capacitation, that permit a spermatozoon to fertilize an oocyte (requires an assortment of signal transduction cascades) 
7. Release from oviductal epithelial cells and passage to the vicinity of the oocyte at the isthmic ampullar junction of the oviduct (requires a coordinated spermatozoal-release mechanism, hyperactivated motility, and probably chemotaxis)  
8. Penetration through the extracellular matrix of the oocyte cumulus (possibly mediated by  hyperactivated motility and redistribution/unmasking of surface-associated hyaluronidase, as the cumulus matrix is rich in hyaluronic acid)  
9. Binding to the zona pellucida, a highly glycosylated protein matrix surrounding the oocyte (appears to involve specific affinity between spermatozoal surface molecules and the zona  pellucida components)  
10. Acquisition of the acrosome reaction, a regulated form of exocytosis (requires reorganization of the outer acrosomal and overlying plasma membranes necessary for fusion and vesiculation) 
11. Penetration of the zona pellucida (release of acrosomal proteins with enzymatic activity is required for this event to occur) 
12. Binding and fusion with the oolemma (requires specific region-dependent molecular interactions)  
13. Dispersion of nuclear contents (requires specific fusogenic alterations of the lipid membranes of the spermatozoon and oocyte)  
14. Oocyte activation (a spermatozoon-derived factor is required for activation of the oocyte and embryonic development).  
15. Pronucleus formation (requires decondensation of the highly compact spermatozoal nucleus) 
16. Organization of the mitotic spindle after pronuclear formation (requires contribution of proximal  centriole from the spermatozoon)  

After viewing this lengthy list of functions, one becomes quite appreciative of the highly complex and  specialized features of a spermatozoon. In fact, the biochemical and biophysical features are so  sophisticated that many of the cellular and molecular mechanisms remain unresolved to this day.  

From Whence They Came  

The life of a spermatozoon begins within the testes, unless, of course, one wishes to consider the  embryonic origin of the primordial germ cells. While endocrine, paracrine, autocrine, cryptocrine, and  lumicrine factors are key orchestrators of testicular function, emerging information is revealing a  multitude of subcellular, molecular-mediated events that “cloud” our understanding of the events that  actually occur within the testes. As with any area of study, the more learned we become about a topic,  the more queries surface that require additional clarification. Such is the case with testicular function.  Without question, a thorough understanding of testicular function will require a keen appreciation of the  mechanisms by which genes and gene products are expressed and repressed. To more fully understand  what makes the testes tick, we must capitalize on the powerful molecular tools that have been developed  in this capacity. Most of these types of studies are conducted in specimens from humans and laboratory  animals, so it will be important to test the relevance to the horse.  

Spermatogenesis  

Spermatozoal production (i.e., spermatogenesis) is an extremely complex process that involves germ cell proliferation, germ cell differentiation, and, paradoxically, programmed germ cell death (termed  apoptosis). This lengthy process, specifically 57 days in length for the stallion, is controlled by a vast  array of messengers acting through endocrine, paracrine, and autocrine pathways. Spermatogenesis not  only involves transformation of undifferentiated diploid germ cells into highly differentiated and  specialized haploid spermatozoa, but it also involves profound transcriptional modifications within the  cells. As a partial list, the finished product of spermatogenesis has: 1) a 1x chromosomal complement that is profoundly repackaged, 2) a newly formed and intricately designed flagellum, 3) the biogenesis  of a highly complex secretory vesicle, the acrosome, and 4) retention of some mRNA (in a largely  depleted cytoplasmic package) that likely has implications in fertilization and post-fertilization events.  

Spermatogenesis occurs within the seminiferous tubules. Both ends of these highly-coiled tubules open  directly into the rete testis, such that the products (both cellular and non-cellular) of the seminiferous  tubules are excreted into the rete testis and delivered to the excurrent duct system (e.g., the epididymis  and ductus deferens). The numerous and tortuous seminiferous tubules, with a combined average length  of 2400 meters per testis in the stallion, are comprised of an epithelial wall, termed the seminiferous or germinal epithelium, and a lumen. The seminiferous epithelium consists of germ cells in various steps of  development intermingled with Sertoli cells that serve to provide structural support and a nurturing  source to the germ cells. The Sertoli cells are anchored to the basement membrane, and extend to the  lumen, of the seminiferous tubules. The seminiferous tubules are bordered by peritubular myoid cells  (myofibroblasts) that, through peristaltic contractions, aid in evacuation of luminal contents into the rete  testis. These myoid cells are also considered to be involved in paracrine signaling events.

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