Evolutionary adaptations of spermatogenesis and testicular structures – spermatogonial stem cell systems, reproductive endocrinology and sperm competition

Male reproduction is challenged by evolution with regard to the formation of „successful” gametes. Amongst these challenges two are most relevant - sperm competition and spermatogonial stem cell (SSC) systems.
While the concept of sperm competition is generally accepted only little is known whether and if by which mechanisms it is reflected in spermatogenesis as such. In principle, three possible mechanisms (or a combination of those) can be suggested: For the sperm, a successful fertilization of the egg occurs when it has won the race. For this purpose, the progressive motility of sperm is crucial, which depends on the number of mitochondria, and the performance of the flagellum, i.e. the length of the sperm. Furthermore, the sheer number of available sperm is a logical determinant which is correlated with the amount of seminiferous epithelium and / or with the rate of sperm production (cycle length).We are interested in the question how sperm competition in testes of primates and birds and have been able to show that there are different adaptations to this challenge in these taxa. While competitive conditions are actually reflected in primates only in relative testes size, birds adjusted the ratio of seminiferous to interstitial tissue and produced sperm of different lengths. Preliminary data also indicate that the duration for the production of mature gametes (spermatogenic cycle length) can vary. The germ line function should also be adjusted for short or long reproductive periods during life. For example, a mouse reproduces only during 1.5 years; monkeys, however, are for fertile many years, increasing the risk of injury or infection. This means that the mission of providing the highest possible number of fertile gametes has to be balanced against maintaining an intact germ line over time. One of the relevant adaptations are different SSC systems, which can be direct (mouse) or progenitor buffered (primates). Our comparative analysis of the organization of the testicular tissue and the endocrine regulation of male reproduction in primates revealed so far unknown findings: On the cellular level and concerning developmental processes, the testicular tissue of New World monkeys corresponds to that of Great Apes (including humans), but differs from Prosimians and Old World monkeys. This could be due to a clonal size and synchrony of SSC derivatives. In contrast, the reproductive endocrine system is very different in New World monkeys from other primates, as luteinizing hormone (LH) is missing and its receptor is mutated, here the Old World monkeys more closely resemble the human situation, via an identical LH / CG - LH receptor system. Thus New World monkeys are a model for research on cellular composition and development of the testis, Old World monkeys, however, should be considered when analyzing hormonal regulation.





Left: Comparative Analysis of primate spermatogenic efficiency proved all monkey species analyzed to exhibit similar rates of sperm production. This indicates sperm competition in monkeys to be reflected only in relative size of testes. Right: New World monkeys have a mutated LHR and developed a reproductive endocrinology dependent on CG, as LH was lost during evolution.

Experimental approaches for translational andrological research

A main topic of male reproductive research is fertility and infertility. In recent decades, research has especially focussed on pathophysiologies that cause infertility, as well as on options for providing a reliable male contraception. To understand the physiology of both of these, translational approaches, i.e. animal experimental work, are necessary. Analyzing knockout mouse models, we have studied different gene defects, such as the CREM and Pax8 deficiency and track genetic causes of infertility currently in a mouse model of the Klinefelter syndrome. Another important topic of andrology aims at providing a reliable male contraception on a hormonal basis which we also investigated. We characterized the substances suggested (testosterone esters and gestagens) in their effect on non-human primates, marmosets and macaques (the model of choice for these studies as results from rodent studies are not or only partially transferable), and assessed the NHPs terms of their suitability for such studies. It was an important finding that New World monkeys, due to their specific endocrine regulation, appear inappropriate for studies on hormonal contraception and should not be used. The preservation of the male germ line by transplantation of germ cells or testicular tissue fragments is also a focus of our research. Besides being able to understand the testicular functions better, methods of germ line transplantation also offer options for preclinical developments. We conducted some xenologous and autologous transplantation studies, in which we demonstrated that the transfer of results obtained in rodents is not readily possible to man. Originally developed as a method for the generation of transgenic animals, later adapted also for fertility preservation in child patients suffering from malignancies, the technique might become also an option to maintain male germ lines of endangered species.

In vitro differentiation of the male germ line – maturation of male gametes in a petri dish

Spermatogenesis is an extremely complex process. For over a century it has been tried to mimic maturation of germ cells in cell and organ cultures in order to understand the cooperation and communication between the somatic cells of the testis and the germ line and to achieve haploidization and sperm production from immature germ cells into spermatozoa in vitro. Despite decades of research, these approaches have remained largely experimental. In recent years, however, important principles were discovered enabling the use of new culture systems, which may be useful to differentiate mammalian sperm in vitro. Although limited in terms of efficiency, recently living mice were generated by assisted reproduction technology with sperm derived from tissue cultures for the first time by a Japanese group. In cultures with mixtures of isolated testicular mouse cells we could obtain morphologically differentiated spermatozoa after a few weeks, differentiated from diploid pre-meiotic cells placed in culture before. We used either a novel matrix-based (Soft Agar Culture System (SACS), methylcellulose Culture System (MCS), or a collagen scaffold as three-dimensional culture systems, which can also be found in regenerative medicine applications.

Rodent testicular cells colonize a collagen scaffold in vitro. A) 2 hours after seeding of a single cell suspension the cells are found attached to the surface of the scaffold. B)  After one day in culture wide parts of the scaffold contain testicular cells and first aggregation into small clusters is seen. C) On day 3 of culture clusters are composed of various cells types showing first signs of reassembly.

Klinefelter Syndrome as an example for the consequences of a disturbed sexchromosomal balance: the 41, XXY* mouse model

Klinefelter syndrome (KS: sex-chromosomal aberration, karyotype 47, XXY, incidence of approximately 1: 600) is one of the main complex genetic disorders in men and can be seen exemplarily for effects of a disturbed sexchromosomal balance. It is caused by a non-disjunction of the sex chromosomes during meiosis in the parental generation. The condition results in a heterogeneous phenotype of variable severity. Amongst its prominent features are infertility, hypogonadism, gynecomastia, cardiovascular problems, disturbed bone metabolism, diabetes, and cognitive deficits. In all patients, the progressive loss of germ cells and hypergonadotropic hypogonadism is common. Mouse models of the strain B6Ei.LT-Y* have been successfully established at the CeRA in the last years and resemble remarkably well the human KS phenotype. We could demonstrate that body proportions of our animal model for KS, 41,XXY* male mice, are altered, that they develop hypergonadotropic hypogonadism in adulthood, and are infertile due to progressive germ cell loss. We also found these male mice to exhibit disturbed memory recognition. Leydig cell function or maturation had been suggested for years and by several groups to be disturbed resulting in insufficient response to endocrine stimulation due to LH receptor dysfunction or steroidogenic failure. Alternatively, Leydig cell hyperplasia was discussed as a putative consequence of incomplete maturation. However, we recently questioned these concepts by demonstrating in our mouse model Leydig cells to be mature and (hyper)active and that in KS patients intratesticular T (ITT) values were not different from control patients but they appear to exhibit altered testicular blood flow. These exciting results put forward a new concept on the origin of hypergonadotropic hypogonadism but also demonstrate the validity of our mouse model, with its translational consequences for our understanding of KS patients. The availability of our mouse model, the option to analyze gene expression patterns and somatic cell responses and the close cooperation between clinics and basic science in our Centre puts us in the privileged situation not only to study aspects of the mechanisms by which e.g. escapee genes provoke a complex phenotype but also to directly translate those findings into a clinical context as we did during the previous project periods.

Figure (adapted from Wistuba 2010)
Characterization of 41, XXY* male mice: Metaphase fluorescence in situ hybridization identification of heterosomes in a 41, XXY* male nucleus counterstained by DAPI. A) The green probe detects the X-chromosomes in the cell arrested in metaphase spread. B) The red probe detects the Y–chromosome. C) the overlay shows the Y-chromosome signals in close association to one X-chromosome. Testicular histology of adult 41, XXY* vs. 40, XY* littermate, haematoxylin staining. D) All seminiferous tubules of the XY* controls exhibited complete spermatogenesis and apparently normal distribution and number of Leydig cells (LC). E) In contrast, 41, XXY* males presented SCO tubules of decreased diameter and Leydig cell (LC) hyperplasia. The bar represents 10 μm. F) The loss of germ cells in adult 41, XXY* males is reflected in significantly reduced testis weight compared to both control. Hypergonadotropic hypogonadism results in G) reduced testosterone (XY* littermate controls vs. XXY*: not significantly different: p = 0.621, C57Bl/6 XY vs. XXY*: significantly different: p = 0.040; XXY*: n = 45, XY*: n= 45, C57Bl/6: n = 33) but H) significantly elevated FSH and I) LH serum levels in XXY* mice compared to both control groups (XXY*: n = 39, XY*: n= 38, C57Bl/6: n = 29). None of the parameters measured differed significantly between the both control groups.