Studies on Testicular Development and Aging in the Common Marmoset (Callithrix jacchus) and the Establishment of Somatic Testicular Cell Lines
by Syed Bilal Hassan
Date of Examination:2025-08-07
Date of issue:2025-10-15
Advisor:Prof. Dr. Rüdiger Behr
Referee:Prof. Dr. Rüdiger Behr
Referee:Prof. Dr. Gregor Bucher
Referee:Prof. Dr. Susann Boretius
Referee:Dr. Christian Roos
Referee:PD Dr. Michael Winkler
Referee:Dr. Ufuk Günesdogan
Persistent Address:
http://dx.doi.org/10.53846/goediss-11565
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Abstract
English
The phenomenon of reproductive ageing in women has been known for a long time and some aspects of this phenomenon are well understood. However, ageing effects in the testes were not recognized for a long time, as aged males can still produce offspring. Only in recent years more and more findings have been made on the male side that the mammalian testis also shows clear signs of ageing, which could also be relevant for the development and health of the offspring. These effects and findings are important for human reproductive medicine and family planning, as the age of fathers has increased significantly in recent decades, particularly in “western” societies. For example, the average age of men at the birth of a child in Germany rose by 3.7 years between 1991 and 2022. Studies on male reproductive ageing are therefore necessary from the epidemiological-psychological level down to the molecular level in order to better understand this phenomenon and assess its effects. In the first part of the present study, the expression pattern of two factors in the testis of the common marmoset (Callithrix jacchus), which is a useful model for human testicular biology, was investigated: p16INK4a and NR2F1. For this purpose, (1) analyses were performed during three stages of postnatal testicular development and (2) studies of adult testes in four age groups. p16INK4a is known as a tumor suppressor protein that plays an important role in cell cycle regulation. It is encoded by the CDKN2A gene. Via cyclin-dependent kinases, p16INK4a inhibits the phosphorylation of the retinoblastoma protein and represses the transition of the cell from G1 to S phase. However, its role in the primate testis has only been initially investigated and not in a cell-type- and development-specific manner. Here we show that p16INK4a is expressed in all developmental stages and age groups of the marmoset testis. The expression of p16INK4a was restricted to the tubular compartment. p16INK4a is first specifically detectable in immature germ cells in the neonatal testis, but then in the adult testis at the protein level only in Sertoli cells. Furthermore, the expression of p16INK4a in the adult testis was age-dependent, with the proportion of Sertoli cells expressing p16INK4a increasing significantly with age. However, this observation does not appear to apply to all primate species. In the common marmoset testis, however, p16INK4a shows a clear development- and age-dependent expression pattern. NR2F1 belongs to the group of nuclear orphan receptors. Its known functions include the regulation of central nervous system development, including the visual pathway. However, the expression of NR2F1 in the mammalian testis is unknown at the protein level and during development and aging; only limited transcriptomic data are available. In newborn testes, NR2F1 is detectable in both the tubular compartment (germ cells) and the interstitial compartment (possibly immature Leydig cells). In juvenile marmosets, expression was mostly restricted to the tubular compartment (germ cells), but a few interstitial cells (possibly Leydig cells) were also stained. In adult testicular tissue, NR2F1 was specifically detectable in spermatogonia. It was shown that NR2F1 expression increases with age, but then decreases in the oldest testes of marmosets. In general, NR2F1 is a novel marker for spermatogonia in the adult testis of the marmoset. In vivo studies in primates, including humans, are often neither ethically nor practically feasible. Therefore, cell-based in vitro models of testicular function play an important role in understanding at least some aspects of gonadal function. Here, immortalized cell lines (as opposed to primary cell cultures) can be a useful substitute for in vivo studies as well as an unlimited resource due to their long-term stability in cell culture and the fact that they do not require further animal invasive procedures. Therefore, in the second part of this thesis I have generated immortalized monoclonal cell lines from adult and juvenile Sertoli and Leydig cells of marmosets. Immunofluorescence and PCR analyses confirmed the identity of one juvenile and one adult immortalized Sertoli and Leydig monoclonal cell line. The identity of the generated cell lines was confirmed by transcriptome analysis for the juvenile Sertoli and both Leydig cell lines, whereas the identity of the adult Sertoli cell line could not be confirmed and the results rather point to a Leydig cell identity. Overall, to our knowledge for the first time, somatic cells from the testis of the marmoset could be immortalized and are available as a resource for future in vitro studies, e.g. for in vitro interaction studies with germ cells or germ cell-like cells. Taken together, the results presented contribute to a better understanding of aspects of aging in the marmoset testis and provide a new resource for animal-free in vitro investigation of primate testicular function.
Keywords: non-human primate; testis; testicular development; testicular aging; p16INK4a; NR2F1; Sertoli cell; Leydig cell; immortalized cell line
