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. The technical aspects of this work may be of interest to labs working on the specific topics of in vitro spermatogenesis for fertility preservation.

Second review:

The authors should be commended for substantial improvement in their manuscript for resubmission.

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.

Overall, the authors addressed most comments and questions from the previous review. The additional data regarding the necrotic area is helpful for interpreting the quality of the cultures.

The authors did not conduct a multiple comparison tests although there are multiple comparisons conducted on for a single dependent variable (Fig 2J, Fig 3F, among many others), however, the addition of this multiple comparison is unlikely to change the conclusions of the paper or the figure and, thus is a minor technical detail in this case.

Author Response

The following is the authors’ response to the original reviews.

We would like to thank you for your thorough review of the manuscript. We have taken all comments into account in the revised version of the manuscript. Please find below our detailed 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 have carefully considered all comments and made the requested revisions to improve the manuscript.

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 suggested, we have reworked 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 under physiological and pathological conditions, and could also be useful for testing the toxicity of cancer therapies, drugs, chemicals and environmental agents (e.g. endocrine disruptors) on the developing testis.

There is a crucial unmet need to optimize the culture conditions for in vitro spermatogenesis. It is important to identify the deregulated molecular mechanisms leading to a decreased in vitro spermatogenic yield. Such results will be of great help to improve organotypic culture conditions. In the present study, we not only uncovered for the first time a failure in adult Leydig cell development, but also an alteration in the expression of several steroidogenic and steroid-metabolizing genes, which could explain the accumulation of progesterone and estradiol and the deficiency of androstenedione in cultured tissues. This hyperestrogenic and hypoandrogenic environment could explain, at least in part, the low efficiency of in vitro spermatogenesis. Furthermore, we show that the addition of hCG (LH homolog) is not sufficient to facilitate Leydig cell differentiation, restore steroidogenesis and improve sperm yield. These data provide valuable information for improving culture conditions. More fundamentally, this culture system could be a useful tool for identifying factors that are essential for the differentiation and functionality of adult Leydig cells during puberty initiation.

Recommendations For The Authors:

This reviewer appreciates that a lot of work was put into this manuscript in terms of experiments, systems, and approaches. However, the manuscript needs significant revision, and in this reviewer's opinion is not appropriate for a broader readership journal. The results seem rather incremental, and the topic is too specialized in its current format.

The manuscript was significantly revised taking into account the reviewer’s comments. In addition, as mentioned above, the development of a model of in vitro spermatogenesis could have wider applications and be of interest to a broader audience.

Comments for improvement, roughly in order of appearance:

1. Abstract - would recommend condensing to hit the main points of the manuscript.

The abstract has been condensed as suggested.

1. Introduction, overall - this is a rather loosely organized list of information that is not synthesized or communicated in a meaningful way. It contains overstatements and lumps together findings from both mice and primates and thus several statements for the actions of these steroid hormones are inaccurate. The authors rely much too heavily upon reviews and need to replace those with a more scholarly approach of carefully reading and citing primary literature.

The Introduction has been reorganized to make it clearer, more synthetic, more meaningful and more accurate. Only findings from rodents are presented. We carefully read the literature and replaced most of reviews by primary literature.

1. Results - this section was extremely difficult to read and comprehend, as it's essentially a laundry list of measurements of mRNAs, steroids, cholesterols, and proteins that go up or down or don't change at multiple ages, both in vitro and in vivo. The section would be improved greatly by an organization with rationale and concluding statements to prepare the reader for the factoid-style data that are presented.

As suggested, the Results section has been improved by an organization with rationale and concluding statements to make it easier to read and comprehend.

1. 47 - is this approach going to both "preserve and restore"? Sounds more like it will allow for the production of offspring, but the other goals are not going to happen from the approach listed in the latter part of that sentence - so not really "fertility restoration" but more of an insurance program that sperm can be produced for ART

Freezing of prepubertal testicular tissue, which contains spermatogonia, is a fertility preservation option proposed to prepubertal boys with cancer prior to highly gonadotoxic treatments. Several fertility restoration strategies, which aim to allow the production of spermatozoa from cryopreserved spermatogonia, are being developed, including in vitro spermatogenesis. This sentence has been rewritten.

1. 62 - specify whether this "decreased expression" is mRNA or protein, and is this because of a loss of Sertoli cells?

“Decreased expression” was replaced by “decreased mRNA levels”. The results we obtained in the cited study (Rondanino et al., 2017) suggest that the decrease in Rhox5 mRNA levels is not the consequence of a change in the proportion of Sertoli cells but reflects an alteration in Rhox5 gene expression. In Figure 6U of the present study, we show indeed that there is no loss of Sertoli cells in organotypic cultures.

1. 66 - what is "the first wave of mouse in vitro spermatogenesis"? Are these cultures from the first wave of mouse in vivo spermatogenesis, or is there a second wave of in vitro spermatogenesis? Please specify

In the mouse, the first entry into meiosis occurs around 8-10 dpp and the first spermatozoa are produced at around 35 dpp: this is the first wave of spermatogenesis which takes place at the onset of puberty. By culturing 6 dpp-old testes for 30 days, our aim is to reproduce in vitro all the stages of this first wave of spermatogenesis, i.e. entry into meiosis, completion of meiosis and spermiogenesis.

In the cited study (Pence et al., 2019), the authors cultured 5 dpp testes for 35 to 49 days and observed a decline in intratesticular testosterone levels in the cultured tissues, i.e. after the end of the first spermatogenic wave, compared to in vivo controls. Our sentence has been rewritten to make it clearer.

1. 78 - is there a difference in T production by Fetal vs Adult LCs? It is this reviewer's understanding that the levels of T around birth in mice (and then a few months after birth in humans) are quite high, similar to adults. So, what are the authors suggesting here by providing the list of expressed genes in these two LC populations?

As mentioned in the Introduction section, 17β-HSD3 – the enzyme responsible for the conversion of androstenedione to T – is not expressed in fetal Leydig cells but is expressed in adult Leydig cells. Therefore, unlike adult Leydig cells, fetal Leydig cells are not capable of synthesizing T.

In the present study, we investigated steroidogenesis but also wondered which types of Leydig cells could be detected under in vitro conditions. It is therefore important to explain to the reader which steroidogenic proteins are expressed by the different Leydig cell populations.

As described in O’Shaughnessy et al., 2002, levels of intratesticular T decline after birth, being very low between 10 and 20 dpp. Then, T levels increase. At 25 dpp, T levels are close to those observed at 1 dpp. T levels increase more than 16-fold between 25 and 30 dpp and then double between 30 dpp and adulthood. Therefore, intratesticular T levels around birth in mice are not as high as in adults, but are about 36-fold lower after birth than in adulthood. It has been shown that in the fetal testis, the conversion of androstenedione produced by fetal Leydig cells is achieved by the adjacent fetal Sertoli cells that express 17β-HSD3 (O’Shaughnessy et al., 2000; Shima et al., 2013). During postnatal development however, Sertoli cells lose the expression of 17β-HSD3 (O’Shaughnessy et al., 2000).

1. 79 -99 - can the authors revise this long list of information to provide a summary of what they are trying to communicate to the reader? What is the intention of this information?

This paragraph has been modified to make it clearer and more synthetic. As different Leydig cell markers are presented in the Results section, it is important to introduce the reader to the different types of Leydig cells, the proteins expressed by these cells and the factors involved in their proliferation and differentiation.

1. 101-2 - replace "involved in" with a more meaningful word - and it is this reviewer's understanding that T has not been shown convincingly to have much of a role in spermatogonial development, at least in mice - that statement is likely true in primates, but not mice; provide primary literature citations to be more precise, rather than a broad review that covers multiple species

“involved in” was replaced by “is essential for many aspects of spermatogenesis, including”. Moreover, we removed “spermatogonial proliferation and differentiation” and provide primary literature citations to be more precise.

1. 105-7 - similar concern for E as for T, above - KO mouse models for ERalpha and beta did not show defects in spermatogenesis as described - not sure what evidence the authors are specifically referring to here - cite primary literature rather than a review on Vitamin D + estrogen

We agree that the question of whether estrogens play a direct role in spermatogenesis was unanswered by the ER null mice. However, estrogens have been shown to be important for the long-term maintenance of spermatogenesis in the ArKO mouse (Robertson et al., 1999) and for the progression of normal germ cell development in the ENERKI mouse (Sinkevicius et al., 2009). This sentence has been reworded and primary literature is cited to be more precise.

1. 113-4 - there is no convincing evidence this reviewer is aware of that the AR is expressed in male germ cells, and therefore T actions on germ cells are indirect, through Sertoli cells and perhaps PTMs; if there is some, this sentence needs a citation showing that

We agree that there is no evidence that AR is expressed in male germ cells and that T acts indirectly on germ cells. This sentence has been rewritten.

1. 114-6 - this is untrue - nowhere in that paper was testosterone or androgen even mentioned!

This reference has been removed. We apologize for this mistake.

1. 116-7 - again, E actions through the ERs are thought to be indirect in the testis, not acting on germ cells; if this is incorrect, please add supportive citations and explain; replace "involved" with a more meaningful word; Rhox5 has a very minor role in spermatogenesis

In contrast to androgen receptors, which are localized in somatic cells, estrogen receptors have been found in most testicular cells, including germ cells. The studies reporting the expression of estrogen receptors in germ cells are cited in the Introduction section. The word “involved” was replaced by “promotes”.

Rhox5 (also known as Pem) has not a very minor role in spermatogenesis. On the contrary, its expression is crucial for normal spermatogenesis and sperm maturation, as loss of Rhox5 in male mice leads to reduced fertility, increased germ cell apoptosis, decreased sperm count and decreased sperm motility (MacLean et al., 2005).

1. 117 - Ref 29 does not support the statement about Rhox5's role in spermatogenesis

The reference (MacLean et al., 2005), supporting the statement about Rhox5’s role in spermatogenesis, was added in the manuscript.

1. 120 - Does FAAH have a protective role in that it is anti-apoptotic? Or just required for some other Sertoli cell function? Should re-word to be more specific.

FAAH (fatty acid amide hydrolase), whose expression is stimulated by estrogens, has been shown to have a crucial role in promoting survival of Sertoli cells by degrading anandamide (N-arachidonoylethanolamine), an endocannabinoid which has a pro-apoptotic activity (Rossi et al., 2007).

The sentence has been reworded to be more specific.

1. 127 - should complete the Introduction with a sentence summarizing what was done and found, for reader clarity

The Introduction has been completed for reader clarity.

1. 136 - misspelled the procedure

Orchidectomy was replaced by orchiectomy.

1. Mice - why use half-day nomenclature for postpartum mice? This is not standard in the literature.

Half-day nomenclature was used due to the uncertainty of the time of birth, which mostly takes place during the night. Since this is not standard in the literature, half-day nomenclature was removed in the entire manuscript.

1. 172-3 - the half-life of RA is very short (<1 hr), and it is light-sensitive. This addition every 8 days means that retinoids are present for a very minimal window of time - are the authors sure retinoids have no requirement elsewhere during spermatogenesis? And in the literature, the measured pulse of RA in the mouse lasts >40 hours (stages VII-IX)...

RA is mandatory for proper spermatogenesis and is needed many times during spermatogenesis (for review, see Schleif et al., 2022): RA is involved in spermatogonial differentiation, pre-meiotic activation and meiotic completion, establishment of the blood-testis barrier and spermiation. In our study, we did not add RA in the culture medium but retinol, the precursor of RA. Indeed, our previous studies have shown beneficial effects of retinol on in vitro spermatogenesis, including an increased production of spermatids with less nuclear alterations and DNA damage (Arkoun et al., 2015; Dumont et al., 2016).

The reason we added retinol (and not RA, which has a very short half-life) in this study and in our previous studies is that it can be oxidized into RA but also be stored in Sertoli cells in the form of retinyl esters for later use. As retinol is photosensitive, handling and storage were performed in tubes covered with aluminum foil, which protects from direct light exposure.

1. 362 - Start the Results section with a broader statement(s) that prepares the readers rather than jumping into specific experiments; it would be helpful for readers to have concluding sentences included as well for readers to navigate the Results section.

As suggested, the Results section has been improved by an organization with rationale and concluding sentences to facilitate reading.

1. 364 - KI67 is a marker of.

Ki67 is widely used as a cell proliferation marker.

1. 367 - replace "involved".

“involved” was replaced by “necessary for”.

1. What intensity thresholds were used to define a cell as positive or negative for a given marker? And there seemed to be no mention of controls - especially no primary antibody controls. This is a significant oversight if these were not done in parallel with every single immunostaining experiment.

We did not apply intensity thresholds. Cells presenting detectable labeling were defined as positive, while unlabeled cells were defined as negative.

Negative controls, performed by omitting the primary antibodies, were of course done in parallel to each immunostaining and are presented in Figure 1A, Figure 2J and Figure 5C. The mention of negative controls has been added in the Materials and methods section.

1. 388 - INSL3 - is this referring to mRNA or protein? Protein nomenclature is used...

INSL3 is here referring to the protein, whose concentrations were measured by radioimmunoassay.

1. 402 - typo.

“expect” was replaced by “except”.

1. 409 - do mRNA levels really "determine the testicular steroidogenic potential"??

This sentence has been reworded: “determine the testicular steroidogenic potential” was replaced by “highlight a potential deregulation of their expression”.

1. 410 - western should not be capitalized.

Western Blot was replaced by western blot in the entire manuscript.

1. 405-28 - this reviewer is underwhelmed by qRT-PCR results for a handful of markers - what is the purpose? The results do not prove anything about the function of the system.

As the differentiation of Leydig cells is not fully completed in organotypic cultures, we wanted to know which actors of the steroidogenic pathway show deregulated expression in vitro in comparison to physiological conditions, and thus which steps of the steroid hormone biosynthesis pathway may be impaired. We found that the expression of several genes encoding steroidogenic enzymes was decreased in vitro, notably that of Cyp17a1, necessary for the conversion of progesterone to androstenedione. Transcript levels of Hsd17b2, encoding an enzyme that converts estradiol to estrone and testosterone to androstenedione, were also decreased at D30.

Our data therefore show that the expression of several steroidogenic genes and steroid metabolizing genes is deregulated in organotypic cultures but we agree that these results do not prove anything about the function of the system.

We then found an accumulation of estradiol and progesterone, a decrease in androstenedione and unchanged testosterone levels in cultured tissues. The elevation in progesterone and the reduction in androstenedione in in vitro matured tissues could arise from the reduced expression of Cyp17a1. In addition, reduced Hsd17b2 transcript levels may explain why estradiol levels remain elevated in cultures while testosterone levels are similar to controls and androstenedione levels are low.

1. How do the authors interpret data gleaned from tissues containing a variably-sized necrotic core?

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

As in our previous publications and recent RNA-seq analyses (Rondanino et al., 2017; Oblette et al., 2019; 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. In order to be able to compare the healthy part of the in vitro matured tissues with in vivo controls, transcript levels were normalized to housekeeping genes (Gapdh and Actb) or to the Leydig cell-specific gene Hsd3b1 while protein levels were normalized to ACTB or to 3β-HSD.

1. 520 - after reading to this point, this reviewer was left confused and wondering why any of this is important to the reader unless that reader specifically works on this topic. The way the data were presented makes it nearly impossible for the reader to keep any of the data in their mind as they read. It's a seemingly endless list of ups and downs of many things under many conditions. What is the point of all of this? How will it advance our understanding of spermatogenesis? Or improve in vitro culture? Or help prepubertal cancer patients? Presumably, that will be explained in the Discussion, but at this point, this reviewer honestly has no idea what this all means. Why is this important??

We have modified the Results section by including rationale and concluding statements to make it easier to read and follow for all readers, not necessarily for those working on this topic.

As mentioned above, the identification of the molecular mechanisms that are deregulated in vitro will give us important insights for the optimization of the culture system. The development of an optimized model of in vitro spermatogenesis could lead to several applications, including improving our knowledge of the regulation of spermatogenesis during pubertal development.

In this study, our main findings are that the differentiation of the adult Leydig cell lineage, steroid biosynthesis, metabolism and signaling are altered in organotypic cultures, leading to a hyperestrogenic and hypoandrogenic environment. In addition, we show that the presence of an LH homolog, known to be critical to adult Leydig cell differentiation and to stimulate steroidogenesis, does not rescue the expression of adult Leydig cell markers and of several steroidogenic genes, steroid metabolizing genes and steroid target genes. Other factors required for Leydig cell maturation and functionality will have to be tested in the future on cultured testicular tissues. Improvements to this in vitro maturation procedure in animal models may be useful for future cultures of human testicular biopsies, although we are aware that more work needs to be done before prepubertal cancer patients can benefit from this in vitro maturation approach.

1. 619-20 - this sort of summarizes this reviewer's overall opinion of the manuscript. Not much seems to have been learned here that would justify publication in a broad readership journal like eLife. More work needs to be done to provide that sort of meaningful advance. The current work, with considerable re-writing to improve accuracy and clarity, is much better suited to a specialty journal where others who are working on this specific topic will appreciate its value.

We have carefully considered the reviewer’s comments and modified the manuscript to improve accuracy and clarity. We understand the reviewer’s point of view, but we believe that this work may be of interest not only to labs working on fertility preservation and restoration, but also to those working on puberty initiation, germ and somatic cell maturation, steroidogenesis under physiological and pathological conditions, and on the effect of cancer therapies, drugs, chemicals and environmental agents (e.g. endocrine disruptors) on the developing testis.

As mentioned above, we not only uncovered for the first time a failure in adult Leydig cell development, but also an alteration in the expression of several steroidogenic and steroid-metabolizing genes, which could explain the accumulation of progesterone and estradiol and the deficiency of androstenedione in cultured tissues. This hyperestrogenic and hypoandrogenic environment could explain, at least in part, the low efficiency of in vitro spermatogenesis. Furthermore, we show that the addition of hCG (LH homolog) is not sufficient to facilitate Leydig cell differentiation, restore steroidogenesis and improve sperm yield. These data provide valuable information for improving culture conditions. More fundamentally, this culture system could be a useful tool for identifying factors that are essential for the differentiation and functionality of adult Leydig cells during puberty initiation.

1. Why are the figures repeated at the end of the manuscript?

During the submission process, our bioRxiv preprint (which contains the figures) was merged with the same but higher quality figures.

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 (Rondanino et al., 2017; Oblette et al., 2019; 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 histological 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 (Figure 8-figure supplement 1). 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 previously determined that 0.75 mm3 was the most appropriate size for mouse in vitro spermatogenesis (Dumont et al., 2016). We do not exclude at all 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 (Rondanino et al., 2017; Oblette et al., 2019; 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. In order to be able to compare the healthy part of the in vitro matured tissues with in vivo controls, transcript levels of the selected genes were 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 the cultured 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.

Images showing mouse testicular tissues cultured for 16 and 30 days are presented in Figure 8-figure supplement 1. The cultured tissues resemble those shown by Sato et al., 2011. As mentioned above, the central necrotic area represents on average 16-27% of the explants. No significant difference in the area of the necrotic region was found between the two culture time points.

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

Bonferroni corrections are used when multiple comparisons are conducted. As mentioned in the Materials and methods section, multiple comparisons were not made in this study. Indeed, the non-parametric Mann-Whitney test was used to compare two conditions: in vitro vs in vivo (D16 FT vs 22 dpp, D16 CSF vs 22 dpp, D30 FT vs 36 dpp, D30 CSF vs 36 dpp, D30 FT + hCG vs 36 dpp, D30 CSF + hCG vs 36 dpp), cultures of fresh vs frozen tissues (6 dpp vs 6 dpp CSF, D16 FT vs D16 CSF, D30 FT vs D30 CSF, D30 FT + hCG vs D30 CSF + hCG) and cultures with vs without hCG (D30 FT + hCG vs D30 FT, D30 CSF + hCG vs D30 CSF). These comparisons were added in the Materials and methods section.

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 3-HSD 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. For CYP17A1 however, 3B-HSD was used as housekeeping instead of ACTB (Figure 2G).

No correlation was found between the number of Leydig cells per cm2 of testicular tissue shown in Figure 1 and Hsd3b1 mRNA levels presented in Figure 2. However, this result was expected since on the one hand the number of Leydig cells per cm2 was determined in the peripheral region of one tissue section whereas on the other hand Hsd3b1 transcript levels were measured in the entire peripheral region of the cultured fragments. The correction factor used for the analysis of genes expressed in Leydig cells present in the healthy part of the cultured tissues was therefore the Leydig cell selective marker Hsd3b1, as previously described (Cacciola et al., 2013).

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.

RT-qPCR and western blot analyses show that both Cyp17a1 mRNA levels and CYP17A1 protein levels are decreased in organotypic cultures at D30. However, we agree that such a decrease is not visible in immunostaining. No autofluorescence of Leydig cells could be observed in the negative controls (Figure 2J).

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 levels of the Sertoli cell-specific gene Dhh are not reduced in organotypic cultures. We also previously found that the expression levels of the Sertoli cell-specific gene Amh were not reduced in in vitro matured testicular tissues (Rondanino et al., 2017). Moreover, we have recently shown that Sox9, encoding a testis-specific transcription factor, is expressed in organotypic cultures (Dumont et al., 2023). Our recent transcriptomic analysis also revealed that the transcript levels of the pro-male sexual differentiation marker Sry and of the Sertoli cell-specific gene Dmrt1 remained unchanged in organotypic cultures compared to in vivo controls (Dumont et al., 2023). In addition, no increase in the mRNA levels of the female sex-determining genes Foxl2 and Rspo1 was found in vitro (Dumont et al., 2023). However, we cannot rule out that in vitro cultured testes are becoming more female-like as the expression of Hsd17b3, encoding an androgenic enzyme, is reduced (this study) while the expression of the feminizing gene Wnt4 is upregulated (Dumont et al., 2023).

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?

In the present study, abundant CYP19 expression was indeed found at 6 dpp. However, the expression of this enzyme was not measured between 6 dpp and D16. Therefore, we cannot be sure that 6 dpp is the last day with abundant CYP19 expression. We assume that the estradiol synthesized before D16 may then accumulate within the cultured tissues. In our study, we quantified the transcript levels of Sult1e1, encoding an estradiol metabolizing enzyme. SULT1E1 is thought to play a physiological role in protecting Leydig cells from estrogen-induced biochemical lesions (Tong et al., 2004). A reduction in Sult1e1 mRNA levels was found at D30 in comparison to in vivo controls, but this may occur earlier during organotypic culture. In addition, decreased transcript levels of Hsd17b2, which encodes an estrogen metabolizing enzyme that converts estradiol to estrone, were found at D30 in this study. We suggest in the Discussion section that elevated estradiol levels in cultured tissues could be a consequence of low Sult1e1 and Hsd17b2 expression. Our recent transcriptomic analyses show that the levels of Cyp1a1, Cyp1b1 and Comt, encoding other estrogen metabolizing enzymes, are unchanged in organotypic cultures (Dumont et al., 2023). To our knowledge, there is no alternative mechanism for estradiol production.

Recommendations For The Authors:

1. The acronyms, PLC, SLC, ILC, ALC, and FLC, become hard to follow. It is recommended to spell out the names.

PLC was replaced by progenitor Leydig cells, SLC by stem Leydig cells, ILC by immature Leydig cells, ALC by adult Leydig cells and FLC by fetal Leydig cells in the entire manuscript.

1. All Figures: Use letters for each bar graph. Difficult to make a connection from text to figure.

A letter was added to each bar graph.

1. Supplemental figure 1: Change "Changement du milieu" to English.

These words were replaced by “Medium change”.

1. Catalog numbers for antibodies are necessary.

The catalog numbers of the antibodies used in this study are presented in Supplementary Table 1.