Lessons learned in andrology: looking at the spermatozoa in context.

Abstract

My career has involved many switchbacks and pivots, but taken together, has given me a unique vision of sperm biology from basic, applied, and clinical standpoints. I feel deeply honored to be asked to share the lessons I have learned in andrology for this series. A task to which I devote considerable time is reviewing papers for a variety of fertility journals. The lessons I present here address some of the knowledge gaps I see frequently in submitted manuscripts. I became an andrologist because I wanted to do research on goats; I grew up on a suburban ranch in Los Angeles where we raised dairy goats. After completing my bachelor's degree in mathematics, I joined the Peace Corps and worked on a Food and Agriculture Organization of the United Nations goat meat production scheme, assisting with applied research. Back in the states, I looked for a school where I could do a master's degree studying goat production. Edward A. Nelson at Cal Poly Pomona had a UNAID Collaborative Research Support Programs grant, looking at reproduction in small ruminants. He needed a graduate student to set up a semen cryobank. I was tasked with this project and figuring out how to freeze goat and sheep spermatozoa. There was no one at Cal Poly freezing spermatozoa at that time, and I had never collected spermatozoa from any species in my life, so I learned by visiting a local bull stud and endless hours in the library reading countless papers. My experience in my master's program taught me that it was possible to begin work in a scientific area without direct assistance from experts in the field, which brings me to my first lesson: This literature extends back to the early 20th century. Although modern research instrumentation was not available at the time, there are outstanding papers with key observations that remain relevant to work being conducted today. Some of the great papers are from cell biology, zoology, or animal science and are not always searchable on MEDLINE and PubMed. Early papers are not as compact as those today; hence, they are rich with observations that are omitted from more modern literature. Because these papers are not always available online, we are often reinventing the wheel in andrology. I have learned that while formulating a research question, it is helpful to find high quality review papers and follow the references back to the classic papers, looking for observations relevant to my research question. In addition to those of my advisors, there were a number of excellent reviews that had great impact on my early career (Mann, 1964; Blandau, 1969; Mazur, 1970; Graham, 1978; Watson, 1981; Yanagimachi, 1981; Saacke, 1982; Bedford, 1983; Moore & Bedford, 1983; Mortimer, 1983, 1994; Quinn, 1985; Hunter, 1988). From working with goat semen specifically, I learned another important lesson that is often overlooked: While the seminal plasma activates sperm motility and contributes important molecules to the sperm membrane and surface, prolonged incubation in seminal plasma damages spermatozoa in some species. In particular, goat seminal plasma contains high levels of phospholipase A2, and spermatozoa left in the seminal fluid for more than a few minutes die rapidly. Although human seminal plasma is much less toxic, although toxicity can be significant in some men (Rogers et al., 1983), affecting sperm function rather than changing the motility observed at semen analysis. One observation I made when I first started research with human spermatozoa was that cryosurvival is poor if the spermatozoa remain in whole semen for more than the time required for liquefaction. At UC Davis, we had our research donors collect specimens at home and leave them in a locker where a technician picked them up. These spermatozoa did not survive freezing and thawing; but when I had the donors collect at our facility, the problem resolved. Many seminal plasma constituents act on the female reproductive tract rather than providing a medium supporting the fertilizing spermatozoa (Overstreet, 1983; McGraw et al., 2014). The academic part of my master's program was a long haul because I was not even a biologist! It required 2 years of undergraduate coursework. Although I had taken probability and statistics as part of my math degree, I had never even done a t-test. A course by Melinda J. Burrill on experimental design got me started in biostatistics and later in my career, Steven J. Samuels taught me more about biostatistics, and I continue to improve my knowledge of experimental design and data analysis by reading texts, papers and courses online. This is one of my current roles in my department, where I give lectures on experimental design and assist faculty and residents with their research. But back to andrology: I occasionally feel like an army of one reminding authors that it is invalid to describe sperm concentration or total sperm counts from groups of men with the mean and standard deviation (or standard error). Sperm concentration is highly skewed, and must either be transformed to normality, or described and analyzed using non-parametric statistics. For displaying the results, a box plot, the median and inter-quartile range or the median and its 95% confidence interval accurately illustrate these non-normal variables. The difference between means in a study is highly influenced by the outliers having high sperm concentration in each group. When the correct statistical methods are used, they sometimes reveal significant differences that were otherwise obscured. After completing my master's degree, I went on to the University of California, Davis, where I initially continued my work with goat semen. While learning objective measures of sperm motility, I met David F. Katz. I was immediately drawn to his work on the biophysics of sperm motility, and that became my doctoral work. I remained in the Overstreet and Katz research group for a total of 13 years, (including 2 more years of undergraduate coursework in systemic physiology and cell biology), completing my PhD with David Katz; conducting postdoctoral work on human spermatozoa cryobiology with James W. Overstreet and with John H. Crowe in the zoology department; and finally becoming an adjunct faculty member. While working on sperm motility I learned that: Although the flagellum does not beat in both directions, forward motility is achieved by the spermatozoa rolling along the axis of progression. Once motility is hyperactivated, and the sperm head is embedded in the zona pellucida, the large flagellar bends and straightened flagellum cause the sperm head to rock in the zona material, appearing to cut knife-like through to the perivitelline space (Fig. 1). A major advantage of working in the Overstreet and Katz group was that the ideas of even the youngest member of the group (e.g., the undergraduate hired to wash glassware), were taken seriously, as was information from every scientific discipline, basic and applied. This resulted in new ideas that challenged old assumptions. The lessons I learned there, and attending Stanley Meizel's weekly journal club, are too numerous to cover here, but the theme is taking a ‘sperm's eye view’ of sperm function in vivo (Katz et al., 1987). With the advent of the assisted reproductive technologies, the focus has largely shifted away from the rich and complex natural history of the fertilizing spermatozoa on its journey from the testis to the oolemma. Although for basic biology experiments, we must focus on isolated cells and molecules, the most important lesson I learned as an andrologist is always consider the spermatozoon in its biological context: The population of spermatozoa capable of achieving fertilization migrate quickly into the fluids of the female reproductive tract; meanwhile, the seminal fluids are diluted by female tract secretions. Human semen (as in other primates, ruminants and rabbits) is deposited in the vagina in close apposition to the cervix, from which spermatozoa migrate into the cervical mucus. In other species (e.g., rodents, dogs, pigs, horses) whole semen is deposited in, or is rapidly drawn into the uterus, from which spermatozoa migrate through the tight uterotubal junction (UTJ), gaining access to the oviduct. Looking at human semen after liquefaction in a specimen cup is not observing how spermatozoa behave at any time during their natural history in the female. After deposition of semen in the female reproductive tract, fertilization-competent sperm do not remain in the lumina of the tract where sperm are most easily collected for study; rather, the fertilizing population moves in the complex milieu at epithelial surfaces. Although we often use cartoons of the female tract showing fluid-filled tubes, no such structures exist. The lumen of the reproductive tract is minimal, while the epithelia have an enormous surface areas with deep glands, crypts, folds, and ciliated surfaces. The fluid in the small luminal spaces is continually refreshed by secretions, flushing out the spermatozoa less able to maintain refuge in mucins at epithelial surfaces. After deposition in the vagina, spermatozoa with strong motility and appropriate surfaces migrate into the cervical mucus, gaining entry to the uterus by swimming along the surfaces of the cervical epithelial cells. A superb study by Mullins and Saacke (1989) used stereomicroscopic and computer reconstruction on serial sections of the bovine cervix to determine the three dimensional structure of sperm migration. Down in those folds and crypts are where you will find the most motile spermatozoa, migrating to the uterus. Spermatozoa reach the oviduct assisted by adovarian uterine contractions present during the periovulatory phase. At this stage, spermatozoa migrate through the UTJ and form ligand-specific attachments to isthmic epithelial cells. Susan S. Suarez, who was Jim Overstreet's postdoctoral trainee when I joined the group, has done brilliant work on this association in multiple species (Suarez, 2008; Hung & Suarez, 2010). She has also shown that the UTJ, isthmus and ampulla of the oviduct are mucus filled. In association with the isthmic epithelial cells, sperm motility slows and remains quiescent for up to several days. Once ovulation occurs, some sperm detach from the isthmus, in part due to acquisition of hyperactivated motility, and ascend to the oviductal ampulla. The spermatozoon is small with minimal cytoplasm and most of its DNA packaged compactly on protamines, unavailable for transcription. Thus, the spermatozoon relies on epithelial cells and their secretions in the male excurrent tract, female reproductive tract, and eventually in the ooplasm to enable its functions. When we remove spermatozoa from their normal niches, we can no longer be sure we are observing the behavior they would display in vivo. A remarkable example showing that very few sperm are required for normal fertility has been reported in some men with hypogonadotropic hypogonadism. When treated appropriately with gonadotropins, spermatogenesis is initiated, and these men can be fertile with much lower total spermatozoa than is considered normal (Burris et al., 1988), as low as 1 million/mL. Clearly these men have a higher proportion of ‘good sperm’. It is important to remember that the few spermatozoa capable of attaining fertilization in vivo are a small fraction of the large, motley population of seminal spermatozoa that we study. Even after careful sperm selection for IVF, many thousands of motile spermatozoa are inseminated per egg, while only a few are believed to be present at the site of fertilization in vivo (Yanagimachi, 2011). In research studies, a measure or treatment effect seen in the evaluated population of spermatozoa may not apply to the spermatozoa competent for fertilization. When heterospermic insemination is used to deposit equal numbers of motile spermatozoa from two highly fertile males, one of the males will fertilize the majority of oocytes (e.g., Overstreet & Adams, 1971; Vicente et al., 2004). This method, first used in the 1950's in mice (Edwards, 1955) has been used extensively in food animal species to compare the fertility of males and spermatozoa treatments. It shows that even in fertile males, there are subtle differences in sperm quality that influence reproductive success. In fact, vigorous motility is not always a good sign. Under various conditions in vitro, including cooling and warming or freezing and thawing, spermatozoa can display highly active motility and undergo the acrosome reaction due to damage to sperm membranes. Modest increases in intracellular calcium promote both high amplitude flagellar bends and acrosomal exocytosis. While media have millimolar calcium concentrations, intracellular calcium is in the nanomolar range. The spermatozoa remain functional for only minutes after completing the acrosome reaction, so premature acrosome reaction is a bad sign if additional sperm function is required. In contrast, there are times in the sperm's natural history when motility is quiescent, as during transport in the epididymis and when stored in the oviductal isthmus. In my reading while writing a review with Jim Overstreet on the natural history of mammalian spermatozoa in the female reproductive tract (Drobnis & Overstreet, 1992), I learned another lesson of which some sperm biologists seem unaware: Rather that providing the ideal environment for each spermatozoon to achieve fertilization, the female reproductive tract impedes most spermatozoa, ensuring that only one spermatozoon reaches and fuses with the oolemma of each oocyte. This is accomplished, in part, by restriction of spermatozoa lacking required surface characteristics, attack by immunocompetent cells, secretion of fluids that flush spermatozoa from the epithelial surfaces, and production of smooth muscle contractions that serve to remove spermatozoa from the female tract. Only a few spermatozoa with appropriate motility and surface characteristics are able to run the gauntlet and reach the oolemma. If the female tract is excessively stringent, or the population of spermatozoa exhibiting the required characteristics is insufficient, fertilization becomes unlikely. Teleologically, it is in the male's interest to produce many spermatozoa and in the female's interest to reduce sperm numbers to exactly one at the site of gamete fusion (Parker, 1984). It cannot be assumed that secretions and cells collected from the lumina of the female reproductive tract will produce environments most favorable to spermatozoa. Just as I was finishing my doctoral work, the AIDS epidemic had become a major focus. Because men could transmit HIV before serum antibodies were detectable, it was apparent that donor spermatozoa must be quarantined, and the donor re-tested for HIV prior to using his specimens. Although human spermatozoa had been cryopreserved for many years with some success, improved methods were desired to increase the feasibility of universal cryopreservation for donor insemination. The NIH released a request for applications on human sperm cryopreservation, and Jim Overstreet and I were awarded one of these grants. When I re-entered the field of sperm cryopreservation after years in basic science, there was renewed appreciation of the membrane lipid composition in cryosurvival: We have known for decades that sperm membranes are organized laterally into domains composed of specific membrane lipids and associated proteins (Fawcett, 1975; Quinn, 1985). From the standpoint of cryobiology, the composition of various domains is crucial as different lipids undergo their phase transition from sol to gel at different temperatures. As spermatozoa are cooled, each lipid undergoes its phase transition and separates laterally into a gel phase region. Packing faults between adjacent gel regions increases the permeability to key substances, notable calcium ions. Membrane proteins, excluded from newly formed gel domains, aggregate and can fail to disperse following warming. These membrane changes cause the phenomenon of ‘cold shock’, which occurs when spermatozoa (and many other cells) are cooled rapidly above the freezing point (Watson & Morris, 1987). Working with John H. Crowe, we used Fourier transform infrared spectroscopy (FTIR) to detect shifts in the –CH2 absorbance peaks that accompany the phase transition of membrane lipids. We determined that the temperature at which spermatozoa of different species undergo cold shock cryodamage, as measured by potassium leakage and loss of motility, is related to the temperature at which membrane lipids undergo the lipid phase transition (Drobnis et al., 1993). In humans, the critical temperature is at about 20 °C and differs between men. Also at this time, a seminar by Roy H. Hammerstedt (Hammerstedt et al., 1990) got me thinking about the sperm glycocalyx: The glycocalyx, a combination of complex glycans largely attached to membrane glycoproteins is some 70 nm thick in mammalian spermatozoa, thicker than that of most cells. Glycoproteins are added to and removed from the sperm surface during its natural history, changing how it interacts with epithelial surfaces and the oocyte vestments. Looking at sperm membrane proteins without consideration of their complex glycosylation can be misleading. I realized the importance of the lateral domains and surface glycoconjugates to sperm capacitation, and incorporated this information into a review of that subject (Drobnis, 1992). As I have since learned, working with Gary F. Clark, the important glycosyl residues on the sperm surface are not just the terminal monosaccharides, but are the complex, arboreal structures spermatozoa use to evade immune surveillance and interact with their environment, including the oocyte investments (Clark, 2014). One of the accomplishments I am most proud of in my career is working with my student, Ted L. Tollner and reproductive primatologist Catherine A. VandeVoort to produce the first offspring from a non-human primate using cryopreserved spermatozoa (Tollner et al., 1990). This was the first species in which I attempted freeze spermatozoa for which cryopreservation techniques had never been developed. In 1994, I left Davis to become a clinical laboratory andrologist in Obstetrics and Gynecology at the University of Missouri, Columbia. One of the most important lessons I learned in clinical andrology was from a seminar by Rebecca Z. Sokol, and it had a profound impact on how I view care of the infertile couple: Ideally, the goal in reproductive medicine should be to treat male factor infertility, allowing the male patient to achieve pregnancies by natural intercourse. As a patient, the man should have his own medical advocate to ensure he is getting the treatment he needs for his medical condition. Life-threatening conditions can present as an abnormal semen analysis (Jarow, 1994; Jequier, 2006; Esteves et al., 2011). The male patient may not be best served if he is seen only by a gynecologist and the first therapy considered for his infertility is an assisted reproductive technology. Due to the financial challenges shared by academic hospitals over the last two decades, I have only intermittently had a laboratory technician. In consequence, I have personally examined countless semen samples during that time, learning in the process: Surprising as it seems, I have reviewed several papers recently in which liquefaction failure was confused with semen viscosity, even when viscosity was the subject of the study. It can be difficult to differentiate these physical properties in the clinical laboratory, particularly because small areas of coagulum can remain once the large gel mass has apparently liquefied. However, there is an easy way to determine if liquefaction is complete: while the molecular mesh causing hyperviscosity is transparent under phase contrast optics, the coagulum is opaque, and the final stages of liquefaction can be readily observed under the microscope. Although the gel fraction is significant in some species (e.g., pigs and horses), this fraction of human semen is rarely discussed in the literature, even in protocols for semen analysis. Though often absent, the gel droplet fraction can account for over a milliliter of the semen volume and is important for at least two reasons, the gel droplets: (i) exclude spermatozoa, thus leading to overestimation when the total sperm count is calculated; and (ii) have high density and will disrupt gradients used for sperm separation. Gel droplets dissolve over time, but not within the time limits required for accurate determination of motility and preparation for cryopreservation or intrauterine insemination. Determination of the gel droplet volume is easily accomplished by centrifuging the whole semen for 1 min at 200 g. The gel volume can then be observed and the supernatant decanted back into the specimen cup for additional processing. For the 187 semen analyses performed in my laboratory in 2014, 37% had gel droplets with a median volume (interquartile range; range) of 0.2 mL (0.1–0.5; 0.1–1.6), and median percentage of gel in the semen volume of 9% (5–14%; 1–35%). In a recent paper, Rupert P. Amann (2009) argued convincingly that the total sperm count and total sperm count per hour of abstinence were better measures of male fertility than sperm concentration. I agree that this is true for men with low to medium semen volume, and the total count should always be included in reports of semen quality. However, in contrast to ruminants, in which the semen volume is low and the sperm concentration is relatively high, in humans, the semen volume varies widely and the concentration of spermatozoa in direct contact with the cervix after coitus is important. In the case of men producing large ejaculates with normal total counts, the fertility may be impaired because much of the semen is not retained near the cervix, and this will be reflected by the low sperm concentration but not by the total sperm count. It has been a pleasure to write this account of my lifetime journey learning lessons in andrology. This has been a great opportunity as I was forced to revisit my assumptions, shedding a new light on what I have learned in the course of 35 years as an andrologist. I hope that some of these lessons will be useful to younger andrologists following their dreams.