FSTL3: A Crucial Regulator of Sertoli Cell Proliferation

Lead Research Organisation: Royal Veterinary College
Department Name: Comparative Biomedical Sciences CBS

Abstract

Notwithstanding current world population infertility in humans is on the rise. 1 in 10 couples is infertile and in about a third of the cases, the defect arises from the male. Testicular defects are among the principal causes of reproductive deficit in males. Perhaps due to environmental effects male infertility in animals is also on the rise. This has most likely contributed to the reduction in numbers of many animal species in the wild. Understanding mechanisms that regulate testicular development and function is therefore essential for development of therapeutic strategies to alleviate male infertility disorders.

The two major functions of the testis are to produce testosterone and sperm throughout the reproductive life of the species. Generally this begins at puberty when testicular size increases. Increased testicular size is an indicator of active generation of sperm and testicular function. There is, however, a need for age-related waning of testis function so that mutations accumulated with age are not propagated in the population. For seasonal breeders an additional requirement is the cyclical increase and decrease of the testis with the beginning and end of each breeding season. Sertoli cells (SC) in the testis allow for the duplications and development of the cells that give rise to sperm and generally the total number of germ cells produced depends on the number of SC in a testis. This number is fixed very early in life and normally does not change with age.

What is not clear is how the number of SC is determined and whether SC numbers in a testis can be increased later on in life. It is also unclear how testis size reduction is controlled in an age or season dependent manner.

We have found that a natural cell product, follistatin-like 3 (FSTL3) might be crucial in regulating testicular development. Our findings in genetically modified mice lacking FSTL3 in all cells show increased testis size, and a lack of testis size reduction with age. Also, within the testis there is an increase in SC numbers and related increase in cells that give rise to sperm. We, therefore, hypothesise that testicular FSTL3 action is essential for limiting testicular size and age-related decline in testicular function.

There are only two other mouse models with increased testis size, but neither of these have a block in age-dependent reduction of testicular size. Our FSTL3 deletion mouse model, therefore, allows us to ask several crucial questions regarding testis development and function. Here we will first determine whether SC multiplication can be increased by reducing FSTL3 in mice and in tissue culture cells. We will then investigate whether SC numbers, can be increased by deleting FSTL3 only in the SC. This will demonstrate whether SC numbers are controlled by local FSTL3 expressed in the SC or whether there is a systemic requirement for FSTL3 production. Furthermore, we will address whether removing FSTL3 after puberty increases SC numbers. This will demonstrate whether SCs can multiply beyond the first few days after birth, therefore providing the possibility of a therapeutic strategy of inducing SC growth and fertility in infertile males with limitingly small testes. Finally, we will identify which genes and cellular signalling pathways FSTL3 may influence to limit testicular size and help achieve reduction of testis size with age. Our research will, therefore, identify how FSTL3 regulates testis development and function and helps limit reproduction with age. We will be identify the molecular mechanisms of these actions, whether FSTL3 dependent SC number regulation is achieved through its expression solely in SC and whether SC can be induced to grow, by removing FSTL3 at any time after birth. The contributions from this work to our understanding of the processes underlying testis development may support development of preventative and therapeutic approaches to dealing more effectively with male infertility.

Technical Summary

Follistatin-like 3 (FSTL3) is a secreted glycoprotein that binds and inhibits a subset of transforming growth factor beta (TGFbeta) ligands, including activin. To reveal its physiological roles we generated FSTL3 gene-deleted mice (FSTL3 KO). FSTL3 is strongly expressed, like activin, in the testis. We have recently shown that FSTL3 gene deletion increases testicular size, primarily via the increase in Sertoli cell numbers, and blocks age-related testicular size regression in these mice. Here we will test the hypothesis that testicular FSTL3 expression is essential for limiting Sertoli cell number and age-related decline in testicular function. We will first ascertain whether there is continued Sertoli cell proliferation in the absence of FSTL3. Also, whether this increased proliferation affects the differentiation status of Sertoli cells. Further, using Sertoli cell-specific, adult-induced or global FSTL3 knockout mice we will elucidate the roles of FSTL3 in Sertoli cell and germ cell proliferation, key determinants of the maintenance of testicular function. Specifically we will investigate whether 1) FSTL3 action in Sertoli cell alone is sufficient for limiting Sertoli cell numbers and testicular size or whether there is a requirement of a systemic FSTL3 effect; 2) Sertoli cell proliferation can be induced beyond the early post-natal period. We will also identify the signalling pathways and gene transcription events, influenced by FSTL3, that are crucial for maintaining testicular size in the FSTL3 KO and conversely those that are essential for age-dependent testicular regression. Our findings will define the processes underpinning the regulation and maintenance of testicular size during adulthood and ageing, and will support development of preventative and therapeutic strategies for testis-dependent reproductive disorders including male infertility.

Planned Impact

One of the most common disorders across all species is infertility, which in humans is frequently associated with testicular development and function. About 1 in 10 couples are infertile and infertility is on the rise. This is also true for companion and wild animal species. For humans extensive hormone therapy, in vitro fertilisation etc are common avenues of therapy but these are not always successful and often are detrimental to the emotional wellbeing of the people affected. Effective therapeutic interventions are desperately needed
The proposed work is focused on elucidating the mechanisms by which FSTL3 regulate Sertoli cell proliferation, testicular development, function and age-related reduction of testicular size. A greater appreciation of how Sertoli cell proliferation and related spermatogenesis is controlled will offer routes towards treatment of male infertility arising out of reduced testicular size and function. Hence, the contributions of this work to our understanding of the processes underlying testis development, function and its regulation may support development of preventative and therapeutic approaches to dealing more effectively with male infertility related to testicular dysfunction.
The following groups will benefit:
1. Industry: These results will clearly be valuable to the pharmaceutical and biotechnology industries with interests in novel therapeutics for male infertility. Findings related to maintenance of male fertility in mice might translate into alleviating human infertility
2. Policymakers and government agencies: Indirectly, The NHS, DEFRA and animal health and welfare charities will benefit from insights into testicular health revealed by our studies since loss of testicular function including that in chemotherapy patients is responsible for significant healthcare costs, as well as a factor that contributes to the lowering of quality of life.
3. Society: In the long term, the ultimate beneficiaries of the research will be human, and companion and performance animal patients along with their carers, dependants or owners. Loss of or reduced testicular function leads to severe mental anguish, stress and strongly affects emotional well-being in couples wishing to conceive.
4. Academics: Academics researching in a number of fields across biology, medicine and ageing will benefit, as outlined in the "academic beneficiaries" section of this proposal. The most immediate impact may be for reproductive biology researchers who will gain new insight into the control of postnatal testis development.
 
Description Currently, 1 in 10 couples are infertile and about a third of this is due to male infertility. Notwithstanding the world population, infertility is tremendously deleterious to the emotional wellbeing and the quality of life of couples thus affected. The causes and mechanisms of male infertility are complex and new therapies need identification. Only one spermatozoon is needed to fertilise an oocyte. Yet, mammalian male fecundity depends on the production of a large number of spermatozoa. The number of spermatozoa produced depends on the number of Sertoli cell in the seminiferous tubule. Activin and related TGFb family ligands are among the factors that regulate testicular development and function. Activin action can be regulated by FSTL3, an endogenous glycoprotein that binds and inhibits activin. We have shown that a deletion of FSTL3 in mice (FSTL3 KO) leads to increased testicular size in the adult with concurrent increase in Sertoli and germ cell numbers in the adult. Interestingly, testicular mass regression observed during ageing in wild-type males is prevented in FSTL3 deficient mice. Excitingly, the FSTL3 KO testicular size/body ratio is similar to WT at weaning (3 weeks) but 1.5 fold increased by 17 weeks. This suggests that while the number of Sertoli cells are similar between the two genotypes early in life, with age the Sertoli cell number and hence the germ cell components of the FSTL3 KO mice are increased. To begin to investigate testicular cell proliferation we monitored presence of proteins that allow the identification of cellular proliferation, PCNA in FSTL3 KO and WT testes at 3 and 8 weeks, prior to the onset of the first wave of spermatogenesis and in the adult stage, respectively. While we found similar PCNA expression at 8 weeks between the two genotypes, at 3 weeks FSTL3 KO mice showed significantly increased testicular expression of PCNA compared to WT. Furthermore, while sperm count is similar between the genotypes in adult (38 weeks) mice, FSTL3 KO mice have 8 fold greater sperm count, compared to WT, in aged (91 weeks) mice. Also, sperm count in FSTL3 KO mice does not change between the two age groups. This suggests that spermatogenesis persists in FSTL3 KO mice long after the testis has ceased to generate sperm iin WT mice. Very careful measurement of proliferating cells in the testis during the first three weeks of life and identification of whether Sertoli cells show markers of increased proliferation led to the interesting conclusion that Sertoli cells are not proliferating more significantly in the FSTL3 KO mice, but there are more Sertoli cells generated by differentiation from precursor cells in FSTL3 KO mice. This is a very fine point in understanding post-natal testicular biology. Also, a crucial finding that allows us to ask the next interesting question as to how this differentiation is increased in the absence of FSTL3 and how Sertoli cell precursors are affected by FSTL3 deletion. Importantly, we have also identified transcripts that are altered in the absence of FSTL3 in the testis at 3d and 56d. A large number of cellular pathways are affected by FSTL3 deletion, including those associated with cytoskeletal components, cellular proliferation and cell metabolism. We have also identified a significant number of SMAD-dependent transcription events that are altered in the absence of FSTL. Currently we are analysing these large-scale data to delineate molecular mechanisms that are affected by the deletion of FSTL3 iin order to identify roles of TGFb family ligands in testicular development and function.
Exploitation Route Findings from this research will be used primarily by academics to further our knowledge of testicular biology, natural design and systems to allow for reproductive capacity in males. Greater understanding of these might help alleviate male infertility arising out of testicular development or ageing in the future.
Sectors Pharmaceuticals and Medical Biotechnology