Steroidogenesis and androgen/estrogen signaling pathways are altered in in vitro matured testicular tissues of prepubertal mice

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to compare, from testis tissues at different ages from mice in vivo and after culture, multiple aspects of Leydig cells. These aspects included mRNA levels, proliferation, apoptosis, steroid levels, protein levels, etc. A lot of work was put into this manuscript in terms of experiments, systems, and approaches. However, as written the manuscript is incredibly difficult to follow. The Introduction and Results sections contain rather loosely organized lists of information that were altogether confusing. At the end of reading these sections, it was unclear what advance was provided by this work. The technical aspects of this work may be of interest to labs working on the specific topics of in vitro spermatogenesis for fertility preservation but fail to appeal to a broader readership. This may be best exemplified by the statements at the end of both the Abstract and Discussion which state that more work needs to be done to improve this system.

Reviewer #2 (Public Review):

Preserving and restoring the fertility of prepubertal patients undergoing gonadotoxic treatments involves freezing testicular fragments and waking them up in a culture in the context of medically assisted procreation. This implies that spermatogenesis must be fully reproduced ex vivo. The parameters of this type of culture must be validated using non-human models. In this article, the authors make an extensive study of the quality of the organotypic culture of neonatal mouse testes, paying particular attention to the differentiation and endocrine function of Leydig cells. They show that fetal Leydig cells present at the start of culture fail to complete the differentiation process into adult Leydig cells, which has an impact on the nature of the steroids produced and even on the signaling of these hormones.

The authors make an extensive study of the different populations of Leydig cells which are supposed to succeed each other during the first month of life of the mouse to end up with a population of adult and fully functional cells. The authors combine quantitative in situ studies with more global analyzes (RT-QtPCR Western blot, hormonal assays), which range from gene to hormone. This study is well written and illustrated, the description of the methods is honest, the analyses systematic, and are accompanied by multiple relevant control conditions.

Since the aim of the study was to study Leydig cell differentiation in neonatal mouse testis cultures, the study is well conceived, the results answer the initial question and are not over-interpreted.

My main concern is to understand why the authors have undertaken so much work when they mention RNA extractions and western blot, that the necrotic central part had to be carefully removed. There is no information on how this parameter was considered for immunohistochemistry and steroid measurements. The authors describe the initial material as a quarter testis, but they don't mention the resulting size of the fragment. A brief review of the literature shows that if often the culture medium is crucial for the quality of the culture (and in particular the supplementations as discussed by the authors here), the size of the fragments is also a determining factor, especially for long cultures. The main limitation of the study is therefore that the authors cannot exclude that central necrosis can have harmful effects on the survival and/or the growth and/or the differentiation of the testis in culture. In this sense, the general interpretation that the authors make of their work is correct, the culture conditions are not optimized.

Organotypic culture is currently trying to cross the doors of academic research laboratories to become a clinical tool, but it requires many adjustments and many quality controls. This study shows a perfect example of the pitfall often associated with this approach. The road is still long, but every piece of information is useful.

Reviewer #3 (Public Review):

Moutard, Laura, et al. investigated the gene expression and functional aspects of Leydig cells in a cryopreservation/long-term culture system. The authors found that critical genetic markers for Leydig cells were diminished when compared to the in-vivo testis. The testis also showed less androgen production and androgen responsiveness. Although they did not produce normal testosterone concentrations in basal media conditions, the cultured testis still remained highly responsive to gonadotrophin exposure, exhibiting a large increase in androgen production. Even after the hCG-dependent increase in testosterone, genetic markers of Leydig cells remained low, which means there is still a missing factor in the culture media that facilitates proper Leydig cell differentiation. Optimizing this testis culture protocol to help maintain proper Leydig cell differentiation could be useful for future human testis biopsy cultures, which will help preserve fertility and child cancer patients.

Methods: In line 226, there is mention that the central necrotic area was carefully removed before RNA extraction. This is particularly problematic for the inference of these results, especially for the RT-qPCR data. Was the central necrotic area consistent between all samples and variables (16 and 30FT)? How big was the area? This makes the in-vivo testis not a proper control for all comparisons. Leydig cells are not evenly distributed throughout the testis. A lot of Leydig cells can be found toward the center of the gonad, so the results might be driven by the loss of this region of the testis.

What did the morphology of the testis look like after culturing for 16 and 30 days? These images will help confirm that the culturing method is like the Nature paper Sato et al. 2011 and also give a sense of how big the necrotic region was and how it varied with culturing time.

There are multiple comparisons being made. Bonferroni corrections on p-value should be done.

Results: In the discussion, it is mentioned that IGF1 may be a missing factor in the media that could help Leydig cell differentiation. Have the authors tried this experiment? Improving this existing culturing method will be highly valuable.

Add p-values and SEM for qPCR data. This was done for hormones, should be the same way for other results.

Regarding all RT-qPCR data-There is a switch between 3bHSD and Actb/Gapdh as housekeeping genes. There does not seem to be as some have 3bHSD and others do not. Why do Igf1 and Dhh not use 3bHSD for housekeeping? If this is the method to be used, then 3bHSD should be used as housekeeping for the protein data, instead of ACTB. Also, based on Figure 1B and Figure 2A (Hsd3b1) there does not seem to be a strong correlation between Leydig cell # and the gene expression of Hsd3b1. If Hsd3b1 is to be used as a housekeeper and a proxy for Leydig cell number a correlation between these two measurements is necessary. If there is no correlation a housekeeping gene that is stable among all samples should be used. Sorting Leydig cells and then conducting qPCR would be optimal for these experiments.

Figure 2A (CYP17a1): It is surprising that the CYP17a1 gene and protein expression is very different between D30FT and 36.5dpp, however, the immunostaining looks identical between all groups. Why is this? A lower magnification image of the testis might make it easier to see the differences in Cyp17a1 expression. Leydig cells commonly have autofluorescence and need a background quencher (TrueBlack) to visualize the true signal in Leydig cells. This might reveal the true differences in Cyp17a1.

Figure 3D: there are large differences in estradiol concentration in the testis. Could it be that the testis is becoming more female-like? Leydig and Sertoli cells with more granulosa and theca cell features? Were any female markers investigated?

Figure 3D and Figure 5A: It is hard to imagine that intratesticular estradiol is maintained for 16-30 days without sufficient CYP19 activity or substrate (testosterone). 6.5 dpp was the last day with abundant CYP19 expression, so is most of the estrogen synthesized on this first day and it sticks around? Are there differences in estradiol metabolizing enzymes? Is there an alternative mechanism for E production?

Author Response:

We would like to thank you for your thorough review of the manuscript. We will take all comments into account in the revised version of the manuscript. Please find below our provisional responses to your comments.

eLife assessment

This study reports useful information on the limits of the organotypic culture of neonatal mouse testes, which has been regarded as an experimental strategy that can be extended to humans in the clinical setting for the conservation and subsequent re-use of testicular tissue. The evidence that the culture of testicular fragments of 6.5-day-old mouse testes does not allow optimal differentiation of steroidogenic cells is compelling and would be useful to the scientific community in the field for further optimizations.

Thank you for this assessment. We will carefully consider all comments and make the requested revisions to improve the manuscript.

Public Reviews

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to compare, from testis tissues at different ages from mice in vivo and after culture, multiple aspects of Leydig cells. These aspects included mRNA levels, proliferation, apoptosis, steroid levels, protein levels, etc. A lot of work was put into this manuscript in terms of experiments, systems, and approaches. However, as written the manuscript is incredibly difficult to follow. The Introduction and Results sections contain rather loosely organized lists of information that were altogether confusing. At the end of reading these sections, it was unclear what advance was provided by this work. The technical aspects of this work may be of interest to labs working on the specific topics of in vitro spermatogenesis for fertility preservation but fail to appeal to a broader readership. This may be best exemplified by the statements at the end of both the Abstract and Discussion which state that more work needs to be done to improve this system.

As explained below, we will rework and reorganize the manuscript to make it clearer, more meaningful and more precise. We believe that this work may be of interest to a broader readership. Indeed, the development of a model of in vitro spermatogenesis could be of interest for labs working on the specific period of puberty initiation, on germ and somatic cell maturation and on steroidogenesis during this period, and could even be useful for testing the toxicity of cancer therapies, drugs, chemicals and environmental agents (e.g. endocrine disruptors) on the developing testis.

Reviewer #2 (Public Review):

Preserving and restoring the fertility of prepubertal patients undergoing gonadotoxic treatments involves freezing testicular fragments and waking them up in a culture in the context of medically assisted procreation. This implies that spermatogenesis must be fully reproduced ex vivo. The parameters of this type of culture must be validated using non-human models. In this article, the authors make an extensive study of the quality of the organotypic culture of neonatal mouse testes, paying particular attention to the differentiation and endocrine function of Leydig cells. They show that fetal Leydig cells present at the start of culture fail to complete the differentiation process into adult Leydig cells, which has an impact on the nature of the steroids produced and even on the signaling of these hormones.

The authors make an extensive study of the different populations of Leydig cells which are supposed to succeed each other during the first month of life of the mouse to end up with a population of adult and fully functional cells. The authors combine quantitative in situ studies with more global analyzes (RT-QtPCR Western blot, hormonal assays), which range from gene to hormone. This study is well written and illustrated, the description of the methods is honest, the analyses systematic, and are accompanied by multiple relevant control conditions.

Since the aim of the study was to study Leydig cell differentiation in neonatal mouse testis cultures, the study is well conceived, the results answer the initial question and are not over-interpreted.

My main concern is to understand why the authors have undertaken so much work when they mention RNA extractions and western blot, that the necrotic central part had to be carefully removed. There is no information on how this parameter was considered for immunohistochemistry and steroid measurements. The authors describe the initial material as a quarter testis, but they don't mention the resulting size of the fragment. A brief review of the literature shows that if often the culture medium is crucial for the quality of the culture (and in particular the supplementations as discussed by the authors here), the size of the fragments is also a determining factor, especially for long cultures. The main limitation of the study is therefore that the authors cannot exclude that central necrosis can have harmful effects on the survival and/or the growth and/or the differentiation of the testis in culture. In this sense, the general interpretation that the authors make of their work is correct, the culture conditions are not optimized.

When using the organotypic culture system at a gas-liquid interphase, the central part of the testicular tissue becomes necrotic. As previously reported (Komeya et al., 2016), the central region receives insufficient nutrients and oxygen. In vitro spermatogenesis therefore only occurs in the seminiferous tubules present in the peripheral region. As in our previous publications and recent RNA-seq analyses (Dumont et al., 2023), the central necrotic area was removed so that transcript and protein levels in the healthy part of the samples (i.e. where in vitro spermatogenesis occurs) could be measured and compared with in vivo controls. For histochemical and immunohistochemical analyses, only seminiferous tubules located at the periphery of the cultured fragments (outside of the necrotic region) were analyzed. Steroid measurements were performed on the entire fragments.

The initial material was indeed a quarter testis, which represents approximately 0.75 mm3. No growth of the fragments was observed during the organotypic culture period. We agree with the reviewer that the composition of the culture medium is not the only parameter to be considered for the quality of the culture and that the size of the fragments is also a determining factor. We do not exclude that central necrosis can have harmful effects on the survival and/or the growth and/or the differentiation of the testis in culture. Optimization of the culture medium and culture design (so that the tissue center receives sufficient nutrients and oxygen) will be necessary to increase the yield of in vitro spermatogenesis.

Organotypic culture is currently trying to cross the doors of academic research laboratories to become a clinical tool, but it requires many adjustments and many quality controls. This study shows a perfect example of the pitfall often associated with this approach. The road is still long, but every piece of information is useful.

Reviewer #3 (Public Review):

Moutard, Laura, et al. investigated the gene expression and functional aspects of Leydig cells in a cryopreservation/long-term culture system. The authors found that critical genetic markers for Leydig cells were diminished when compared to the in-vivo testis. The testis also showed less androgen production and androgen responsiveness. Although they did not produce normal testosterone concentrations in basal media conditions, the cultured testis still remained highly responsive to gonadotrophin exposure, exhibiting a large increase in androgen production. Even after the hCG-dependent increase in testosterone, genetic markers of Leydig cells remained low, which means there is still a missing factor in the culture media that facilitates proper Leydig cell differentiation. Optimizing this testis culture protocol to help maintain proper Leydig cell differentiation could be useful for future human testis biopsy cultures, which will help preserve fertility and child cancer patients.

Methods: In line 226, there is mention that the central necrotic area was carefully removed before RNA extraction. This is particularly problematic for the inference of these results, especially for the RT-qPCR data. Was the central necrotic area consistent between all samples and variables (16 and 30FT)? How big was the area? This makes the in-vivo testis not a proper control for all comparisons. Leydig cells are not evenly distributed throughout the testis. A lot of Leydig cells can be found toward the center of the gonad, so the results might be driven by the loss of this region of the testis.

When using the organotypic culture system at a gas-liquid interphase, the central part of the testicular tissue becomes necrotic. As previously reported (Komeya et al., 2016), the central region receives insufficient nutrients and oxygen. In vitro spermatogenesis therefore only occurs in the seminiferous tubules present in the peripheral region. As in our previous publications and recent RNA-seq analyses (Dumont et al., 2023), the central necrotic area was removed so that transcript levels in the healthy part of the samples (i.e. where in vitro spermatogenesis occurs) could be measured and compared with in vivo controls. The transcript levels of the selected genes were of course normalized to housekeeping genes (Gapdh and Actb) or to the Leydig cell-specific gene Hsd3b1.

The central necrotic area was consistent between all samples and variables: it represents on average 16-27% of the explants.

Moreover, we would like to point out that the gonads were cut into four fragments before in vitro cultures. It is therefore the central part of these explants that was removed and not the central part of the gonads. The central part of the gonads was thus included in our analyses.

What did the morphology of the testis look like after culturing for 16 and 30 days? These images will help confirm that the culturing method is like the Nature paper Sato et al. 2011 and also give a sense of how big the necrotic region was and how it varied with culturing time.

This point will be addressed in the detailed responses to reviewers.

There are multiple comparisons being made. Bonferroni corrections on p-value should be done.

This point will be addressed in the detailed responses to reviewers.

Results: In the discussion, it is mentioned that IGF1 may be a missing factor in the media that could help Leydig cell differentiation. Have the authors tried this experiment? Improving this existing culturing method will be highly valuable.

The decreased Igf1 mRNA levels found in the present study are in line with the RNA-seq data of Yao et al., 2017. As mentioned in the Discussion section, the addition of IGF1 in the culture medium led to a modest increase in the percentages of round and elongated spermatids in cultured mouse testicular fragments (Yao et al., 2017). However, the effect of IGF1 supplementation on Leydig cell differentiation was not investigated. The supplementation of organotypic culture medium with IGF1 is currently being tested in our research team.

Add p-values and SEM for qPCR data. This was done for hormones, should be the same way for other results.

p-values and SEM are shown for both qPCR and hormone data.

Regarding all RT-qPCR data-There is a switch between 3bHSD and Actb/Gapdh as housekeeping genes. There does not seem to be as some have 3bHSD and others do not. Why do Igf1 and Dhh not use 3bHSD for housekeeping? If this is the method to be used, then 3bHSD should be used as housekeeping for the protein data, instead of ACTB. Also, based on Figure 1B and Figure 2A (Hsd3b1) there does not seem to be a strong correlation between Leydig cell # and the gene expression of Hsd3b1. If Hsd3b1 is to be used as a housekeeper and a proxy for Leydig cell number a correlation between these two measurements is necessary. If there is no correlation a housekeeping gene that is stable among all samples should be used. Sorting Leydig cells and then conducting qPCR would be optimal for these experiments.

Hsd3b1 was used as a housekeeping gene only to normalize the mRNA levels of Leydig cell-specific genes. Therefore, Igf1 and Dhh transcript levels were not normalized with Hsd3b1 since Igf1 is expressed by several cell types in the testis (Leydig cells, Sertoli cells, peritubular myoid cells) and Dhh is expressed by Sertoli cells.

Regarding western blots, the expression of AR, CYP19 and FAAH could not be normalized with 3bHSD since AR is expressed by Leydig cells, Sertoli cells and peritubular myoid cells, CYP19 is expressed by Leydig cells and germ cells and FAAH is expressed by Sertoli cells. We will review the western blot results for CYP17A1.

As shown in Figure 1B, the number of Leydig cells per cm2 of testicular tissue is not significantly different between the different time points in vivo (6 d_pp_, 22 d_pp_ and 36 d_pp_), in vitro (D16 FT and D30 FT) and between the in vivo and in vitro conditions (22 d_pp_ versus D16 FT, 36 d_pp_ versus D30 FT). Similarly, our data in Figure 2A show that Hsd3b1 mRNA levels are not significantly different between the different time points in vivo (6 d_pp_, 22 d_pp_ and 36 d_pp_), in vitro (D16 FT and D30 FT) and between 22 d_pp_ and D16 FT. However, Hsd3b1_mRNA levels were significantly lower in D30 FT tissues compared to 36 d_pp. We will measure the correlation between the number of Leydig cells per cm2 of testicular tissue and Hsd3b1 mRNA levels, as suggested by the reviewer.

Figure 2A (CYP17a1): It is surprising that the CYP17a1 gene and protein expression is very different between D30FT and 36.5dpp, however, the immunostaining looks identical between all groups. Why is this? A lower magnification image of the testis might make it easier to see the differences in Cyp17a1 expression. Leydig cells commonly have autofluorescence and need a background quencher (TrueBlack) to visualize the true signal in Leydig cells. This might reveal the true differences in Cyp17a1.

This point will be addressed in the detailed responses to reviewers.

Figure 3D: there are large differences in estradiol concentration in the testis. Could it be that the testis is becoming more female-like? Leydig and Sertoli cells with more granulosa and theca cell features? Were any female markers investigated?

We show in the present study that the expression level of the Sertoli cell-specific gene Dhh is not reduced in organotypic cultures. We also previously found that the expression level of the Sertoli-cell specific gene Amh was not reduced in in vitro matured testicular tissues (Rondanino et al., 2017). Moreover, our recent unpublished data show that Sox9, a testis-specific transcription factor, is expressed in Sertoli cells in organotypic cultures. These results suggest that Sertoli cells are not becoming granulosa-like cells and that the testis is not becoming more female-like. Markers of granulosa and theca cells were not investigated.

Figure 3D and Figure 5A: It is hard to imagine that intratesticular estradiol is maintained for 16-30 days without sufficient CYP19 activity or substrate (testosterone). 6.5 dpp was the last day with abundant CYP19 expression, so is most of the estrogen synthesized on this first day and it sticks around? Are there differences in estradiol metabolizing enzymes? Is there an alternative mechanism for E production?

This point will be addressed in the detailed responses to reviewers.