Craig Marc Luetjens

New horizons for in vitro spermatogenesis? An update on novel three-dimensional culture systems as tools for meiotic and post-meiotic differentiation of testicular germ cells

Culture and differentiation of male germ cells has been performed for various purposes in the past. To date, none of the studies aimed at in vitro spermatogenesis has resulted in a sufficient number of mature gametes. Numerous studies have revealed worthy pieces of information, building up a body of information on conditions that are required to maintain and mature male germ cells in vitro. In this review, we report on previously published and unpublished experiments addressing murine germ cell differentiation in three-dimensional (3D) in vitro culture systems. In a systematic set of experiments, we examined the influence of two different matrices (soft agar and methylcellulose) as well as the need for gonadotrophin support. For the first time, we demonstrate that pre-meiotic male germ cells [revealed by the absence of meiotic marker expression (e.g. Boule)] obtained from immature mice pass through meiosis in vitro. After several weeks of culture, we obtained morphologically normal spermatozoa embedded in the matrix substance. Complete maturation relied on support from somatic testicular cells and the presence of gonadotrophins but appeared independent from the matrix in a 3D culture environment. Further research efforts are required to reveal the applicability of this culture technique for human germ cells and the functionality of the spermatozoa for generating offspring.

Physiology of Testicular Function

The testis fulfills two essential functions: production and maturation of the male gametes and synthesis and secretion of the sexual hormones.

Clonal Organization of Proliferating Spermatogonial Stem Cells in Adult Males of Two Species of Non-Human Primates, Macaca mulatta and Callithrix jacchus

Abstract The present study examines the existence of clonogenic patterns in the proliferation and differentiation of spermatogonial stem cells in two species of non-human primates, the marmoset and the rhesus monkey. We developed a novel approach to detect proliferating spermatogonial clones in whole mounts of seminiferous tubules. Dual fluorescence labeling of bromodeoxyuridine and acrosin in conjunction with confocal microscopy allows the description of the clonogenic and spatial arrangement of proliferating spermatogonia at specific stages of the seminiferous epithelial cycle. Cross-sections of paraffin-embedded tissue were labeled by the same approach. For both monkey species we demonstrate the presence of proliferating spermatogonial clones of variable size at specific stages of the cycle of the seminiferous epithelium. Detailed analysis of the rhesus monkey reveals proliferating Apale spermatogonia at stages VII and IX of the cycle of the seminiferous epithelium, and of proliferating B spermatogonia at stages II, IV, VI, and XII. Proliferating Apale spermatogonia at stages VII and IX of the cycle are organized in pairs or quadruplets. B1 spermatogonia appear as quadruplets or eight-cell clones, and B2 spermatogonia as 8- or 16-cell clones. We conclude that spermatogenesis in the rhesus monkey is initiated by two divisions of duplets or quadruplets of Apale spermatogonia: a first division at stage VII, after which the clones of Apale spermatogonia separate, and a second division at stage IX, which leads to clones of B1 spermatogonia as well as pairs and quadruplets of Apale spermatogonia replenishing the seminiferous epithelium to maintain the original size of the A spermatogonial population.

The enhanced pre‐ and postnatal study for nonhuman primates: Update and perspectives

The enhanced pre- and postnatal (ePPND) study design has been developed in response to new scientific knowledge and subsequent guideline changes, that is, ICH M3(R2) and ICH S6(R1). The design changes were basically driven by the experiences obtained during preclinical development of biopharmaceuticals. The ePPND concept typically does not apply to pharmaceuticals. In essence, the ePPND design is a PPND study in which key elements of an embryofetal development (EFD) study are being investigated in newborns and infants rather than in the fetus. The current relevant nonhuman primate model is the cynomolgus monkey. The ICH S6(R1) has reached step 4 during June 2011 and provides detailed recommendations on various parameters and the conduct of an ePPND study. By the time this article is written, it appears that for monoclonal antibodies, the ePPND study is the preferred approach although ICH S6(R1) also leaves options for modified EFD and PPND study concepts. Our data also demonstrate that social housing is feasible for developmental toxicity studies in the cynomolgus monkey model.

Sensitivity of male reproductive endpoints in nonhuman primate toxicity studies: A statistical power analysis

To determine the sensitivity of male reproductive toxicity endpoints in NHPs we performed a power analysis of routine and triggered endpoints using control data from sexually mature Asian and Mauritian NHPs. The power to detect a 50% change from control was 13-30% for male reproductive organ weights, ∼30% for testicular volume, 6-66% for seminal analyses and 10-78% for male hormones. Overall, male reproductive endpoints have poor power (less than 80%) to detect a 50% change from control with a group size of 3 monkeys. Confidently identifying adverse male reproductive effects with these endpoints would likely require specialized study designs with larger group sizes. Triggering of non-routine endpoints in cases where there is special concern for male reproductive toxicity is unlikely to increase sensitivity to detect adverse effects.

Physiologie der Hodenfunktion

Dieses Kapitel stellt die physiologischen Grundlagen fur das Verstandnis der dualen Funktion der mannlichen Keimdruse dar. Die nachfolgend beschriebenen Sachverhalte gelten uberwiegend fur die Situation beim Menschen. Tierexperimentelle Befunde wurden in den Fallen herangezogen, in denen Humandaten nicht vorliegen oder aus ethischen Uberlegungen nicht erhoben werden konnen, und um Konzepte und Theorien verstandlich zu machen. Die Beschreibung der Organisation der Hoden und der physiologischen Grundlagen der Keimzellreifung orientiert sich an den Gegebenheiten im menschlichen Hoden und liefert die Grundlage fur das Verstandnis der endokrinen und der lokalen Regulation der Hodenfunktion. Eine detaillierte Beschreibung der Hodenhistologie findet sich in Kapitel 11. Daran schliest sich die Beschreibung des hypothalamisch-hypophysaren testikularen Regelkreises an, der die „Oberhoheit“ bei der physiologischen Regulation der Hodenfunktion besitzt und ein Regelsystem im klassischen endokrinen Sinne darstellt. Lokale Regulationsmechanismen spielen eine bedeutende Rolle beim „fine-tuning“ der Keimzellreifung.