5th International Andrology Symposium
5th International Andrology Symposium
Shosei Yoshida
Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Correspondence: Prof. Yoshida Shosei PhD Higashiyama 5-1, Myodaiji, Okazaki 444-8787, Aichi, Japan
Spermatogenesis is an evolutionarily conserved but physiologically fragile process that requires the testis to operate within a tightly controlled environment. Most mammals externalize their testes into the scrotum, maintaining a cooler temperature than the abdominal cavity. Failure of testicular descent—cryptorchidism—exposes the testis to higher temperatures and may cause defects in spermatogenesis and male infertility. Yet, it has remained elusive how high temperature affects spermatogenesis, and whether heat is the sole causal factor of spermatogenic defects in cryptorchidism. A recent study from our group, using an ex vivo organ culture system, revealed that temperature elevations of only 1–2 °C induce stage-specific blocks in spermatogenesis. In particular, meiotic double-strand break repair was found to be temperature-sensitive, triggering checkpoint-mediated germ cell elimination. We also showed that heat alone does not fully explain the pathology of cryptorchid testis: While all germ cells but undifferentiated spermatogonia are depleted in an artificial cryptorchid testis model—in which testes are exposed to 38 °C—such histological features were not observed in seminiferous tubules cultured at 38 °C or any other temperature tested. Further, we found that intratesticular retinoic acid (RA) levels decline within 40–48 h following testis translocation without overt cell loss, and that exogenous RA could restore spermatogonial differentiation even under body core temperature. Together, these findings indicate that cryptorchid-associated spermatogenesis defects may result from a combined insult, with high temperature impairing meiosis and spermiogenesis, and with RA depletion compromising spermatogonial commitment. Understanding this dual mechanism will open new translational opportunities, including potential therapies targeting RA metabolism.
Key words Spermatogenesis, temperature adult testis, experimental cryptorchidism
La spermatogenèse est un processus évolutivement conservé mais physiologiquement fragile, nécessitant un environnement testiculaire strictement contrôlé. Chez la plupart des mammifères, les testicules sont externalisés dans le scrotum afin de maintenir une température plus basse que celle de la cavité abdominale. L’échec de la descente testiculaire—cryptorchidie—expose le testicule à une chaleur excessive et peut entraîner des altérations de la spermatogenèse et une infertilité masculine. Les mécanismes précis par lesquels la chaleur perturbe la spermatogenèse, et la question de savoir si la température élevée est l’unique facteur causal dans la cryptorchidie, demeuraient toutefois incertains.
Une étude récente de notre groupe, utilisant la culture organotypique ex vivo, montre que des élévations de seulement 1–2 °C induisent des blocages stade-spécifiques de la spermatogenèse. La réparation des cassures double-brin méïotiques apparaît particulièrement thermo-sensible, entraînant une élimination checkpoint-dépendante des cellules germinales. Nous démontrons également que la chaleur ne suffit pas à reproduire l’histopathologie de la cryptorchidie : alors que tous les types cellulaires germinatifs sauf les spermatogonies indifférenciées sont déplétés dans un modèle expérimental de cryptorchidie (38 °C), ce profil n’apparaît pas dans les tubes séminifères cultivés à 38 °C ou à toute autre température testée. De plus, les niveaux d’acide rétinoïque (AR) intratesticulaire chutent 40–48 h après la translocation testiculaire, avant toute perte cellulaire apparente, et l’apport exogène d’AR restaure la différenciation spermatogoniale même à température corporelle.
Ces résultats indiquent que les défauts de spermatogenèse associés à la cryptorchidie résultent d’une agression combinée : la chaleur perturbe la méiose et la spermiogenèse, tandis que la déplétion en AR compromet l’engagement spermatogonial. La compréhension de ce double mécanisme ouvre des perspectives translationnelles, notamment des thérapies ciblant le métabolisme de l’acide rétinoïque.
Most mammals position the testes in the scrotum, where temperature is maintained below core body temperature [1,2]. This anatomical adaptation is widely thought to protect spermatogenesis by sustaining a cooler milieu. By contrast, some mammals (e.g., cetaceans and proboscideans) lack a scrotum but have evolved alternative testicular cooling strategies [2]. These comparative observations underscore temperature control as a central principle of male germ-cell development.
Our laboratory aims to understand spermatogenesis with a particular emphasis on stem cell regulation in the adult testis. [3-5]. A persistent question is how temperature modulates the balance between spermatogonial stem cell (SSC) self-renewal and differentiation, and how subsequent stages—meiosis and spermiogenesis—respond to small thermal shifts. Cryptorchidism provides a clinically relevant lens for this question but is confounded in vivo by extratesticular factors (endocrine and neural inputs) and the difficulty of precisely measuring or controlling intratesticular temperature [6]. We therefore combined an artificial cryptorchidism model with an ex vivo organ culture system to dissect temperature-dependent steps and to assess whether heat alone accounts for the cryptorchid phenotype.
A guiding observation from artificial cryptorchidism is that after translocating adult mouse testes from the scrotum (~34 °C) to the abdominal cavity (~38 °C), seminiferous tubules become depleted of differentiating germ cells, retaining primarily undifferentiated spermatogonia (Aundiff) alongside Sertoli cells. KIT–positive differentiating spermatogonia are absent in this condition [6]. This suggested—initially—that high temperature may block the commitment of Aundiff to the differentiating spermatogonia. The work summarized here tests that hypothesis, identifies stage-specific thermal sensitivities, and reveals an additional, temperature-linked retinoic acid (RA) deficiency that helps explain the full cryptorchid pathology [7-10].
We performed surgical translocation of testes from the scrotum to the abdominal cavity (adjacent to the liver), after normal adult spermatogenesis had been established. Intratesticular temperature increased from ~34 °C to ~38 °C (directly measured). Within this “extreme” but controlled model, histology consistently showed shrunken seminiferous tubules largely devoid of germ cells beyond Aundiff, consistent with previous reports and mirroring classic reports of cryptorchid pathology [6].
To isolate temperature as a sole controlled parameter and to exclude systemic influences, we employed a long-term gas–liquid interface organ culture of seminiferous tubules (based on an established by Takehiko Ogawa, Yokohama Japan) [11,12]. Cultures at 34 °C supported full spermatogenesis, including undifferentiated and differentiating spermatogonia, pachytene spermatocytes, round spermatids, and elongated spermatids, although being compromised compared with physiological spermatogenesis occurring in vivo. We then adjusted incubator setpoints in 1–2 °C increments to test a narrow temperature window from ~32 °C to 38 °C, scoring outcomes by histology, immunohistochemistry (including KIT and a meiosis marker Sycp3, late meiotic and haploid cell marker Protamine-GFP transgene).
We evaluated meiotic progression (e.g., completion of meiosis I, appearance of haploid cells) and assessed DNA double-strand breaks (DSBs), through chromosome spread methods combined with immunostaining for DSBs (γH2AX) and meiosis machinery proteins (e.g., SCP1, SCP3, RPA2, RAD51, DMC1, and MLH1) and apoptosis proteins (cleaved Caspase 3) [13]. To assess the role of RA signaling, we measured intratesticular RA levels after testis translocation, profiled expression of key metabolic enzymes by qRT-PCR, and tested whether exogenous retinol/RA supplementation could rescue differentiation in the cryptorchid milieu.
Temperature produces stage-specific blocks rather than a single threshold effect [13].
Contrary to our initial expectation of a single “commitment block of spermatogonial stem cells,” we observed a stepwise, stage-specific pattern of thermal sensitivity:
These observations demonstrate that increases of only 1–2 °C can shift the dominant defects from spermiogenesis (block of spermatid elongation) to meiotic arrest, leading to non-uniform thermal sensitivities across germ-cell stages.
High temperature impairs meiotic DSB repair and induces apoptosis on meiotic checkpoint
At 37–38 °C in culture, meiotic cells showed evidence of increased unrepaired DSBs persisting into pachytene stage, likely triggering meiotic checkpoint–mediated elimination of damaged germ cells. This mechanistic link—DSB repair sensitivity to heat and checkpoint activation—accounts for the abrupt pachytene-stage arrest and loss of downstream haploid cells at higher temperatures. [6,13].
Heat alone does NOT cause the full artificial cryptorchid pathology [6,13]. Despite recapitulating substantial defects in meiosis and spermiogenesis at 37–38 °C ex vivo, we found that the organ culture never reproduced the complete depletion of differentiating germ cells as seen in artificial cryptorchidism (i.e., “Sertoli cell–only” with residual undifferentiated spermatogonia and absence of KIT–positive differentiating spermatogonia and more advanced cells). Thus, while heat clearly disrupts meiosis and spermiogenesis, it does not fully explain the cryptorchid testis histology.
Cryptorchid testes rapidly lose intratesticular RA before cell loss
In the artificial cryptorchid model, RA levels decline within 2 days after translocation to the abdomen, before overt cell loss observed in 3–5 days. This early RA depletion occurs while all major cell types—including pachytene spermatocytes, the predominant RA producers via aldehyde dehydrogenase enzymes—are still present. Bulk RNA-seq before/after translocation did not reveal a clear, systematic change in RA-metabolizing enzyme mRNA levels, suggesting that the drop in RA could arise from post-transcriptional mechanisms.
Exogenous RA triggers spermatogonial commitment at body-core temperature
Administration of retinol, an RA precursor, to mice carrying cryptorchid testes could trigger differentiation of undifferentiated spermatogonia into differentiating spermatogonia. This occurred under body-core temperature (~38 °C), indicating that high temperature does not prevent spermatogonial commitment per se if adequate RA signaling is available.
Our data indicate the pathophysiology of artificial cryptorchid testis involves multiple causes:
The fine granularity of thermal sensitivity (1–2 °C steps) argues that spermatogenesis is buffered only within a narrow thermal optimum, highlighting distinct thermal liabilities of cellular programs (chromosomal synapsis formation, recombination, checkpoint control). The observation that RA levels fall quickly without corresponding mRNA changes for RA metabolic enzymes points to non-transcriptional control of RA homeostasis potentially involves enzyme kinetics and other mechanisms.
This study may provide some practical implications for human cryptorchidism. In particular, targeted modulation of RA metabolism or RA delivery may help preserve or restore some spermatogenesis. However, from a practical perspective, patient will receive orchiopexy before spermatogenesis proceeds upon puberty. In addition, the artificial cryptorchid mouse model is an extreme and controlled setting, making translation to human pathology require caution.
Spermatogenesis exhibits narrow thermal tolerance with distinct, stage-specific sensitivities: spermiogenesis is compromised near 36 °C, meiosis fails by ~37-38 °C through heat-sensitive DSB repair and meiotic checkpoints. Heat alone does not account for the full cryptorchid pathology. Instead, rapid decrease in RA concentration following testis translocation prevents spermatogonial commitment, producing the characteristic depletion of differentiating germ cells. Exogenous RA rescues this commitment step at body-core temperature, pointing to RA metabolism as a potential translational target. We propose a dual-insult mechanism in cryptorchidism: temperature deranges meiosis/spermiogenesis, while RA deficiency arrests spermatogonial differentiation.
a) Ethics Approval and Consent to Participate Investigations were carried out in accordance 326 with the Declaration of Helsinki of 1975, revised in 2008.
b) Consent for publication Not applicable
c) Availability of data and supporting material Not applicable
d) Competing interests Author/s declare that they have no competing interests
e) Funding none
I thank Kodai Hirano, laboratory members in Department of Germ Cell Biology, National Institute for Basic Biology in Okazaki, Japan, and collaborators including Takehiko Ogawa (Yokohama City University) for the organ culture platform. Portions of this work have been published, while the RA components are unpublished and currently under investigation.
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