Full Spermatogenesis Research Articles

Establishment and Characterization of Testis Organoids with Proliferation and Differentiation of Spermatogonial Stem Cells into Spermatocytes and Spermatids.

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility.

Identification of two hidden clinical subgroups among men with idiopathic cryptozoospermia.

Are there subgroups among patients with cryptozoospermia pointing to distinct etiologies? We reveal two distinct subgroups of cryptozoospermic (Crypto) patients based on testicular tissue composition, testicular volume, and FSH levels. Cryptozoospermic patients present with a sperm concentration below 0.1 million/ml. While the etiology of the severely impaired spermatogenesis remains largely unknown, alterations of the spermatogonial compartment have been reported including a reduction of the reserve stem cells in these patients. To assess whether there are distinct subgroups among cryptozoospermic patients, we applied the statistical method of cluster analysis. For this, we retrospectively selected 132 cryptozoospermic patients from a clinical database who underwent a testicular biopsy in the frame of fertility treatment at a university hospital. As controls (Control), we selected 160 patients with obstructive azoospermia and full spermatogenesis. All 292 patients underwent routine evaluation for endocrine, semen, and histological parameters (i.e. the percentage of tubules with elongated spermatids). Moreover, outcome of medically assisted reproduction (MAR) was assessed for cryptozoospermic (n = 73) and Control patients (n = 87), respectively. For in-depth immunohistochemical and histomorphometrical analyses, representative tissue samples from cryptozoospermic (n = 27) and Control patients (n = 12) were selected based on cluster analysis results and histological parameters. This study included two parts: firstly using clinical parameters of the entire cohort of 292 patients, we performed principal component analysis (PCA) followed by hierarchical clustering on principal components (i.e. considering hormonal values, ejaculate parameters, and histological information). Secondly, for histological analyses seminiferous tubules were categorized according to the most advanced germ cell type present in sections stained with Periodic acid Schif. On the selected cohort of 39 patients (12 Control, 27 cryptozoospermic), we performed immunohistochemistry for spermatogonial markers melanoma-associated antigen 4 (MAGEA4) and piwi like RNA-mediated gene silencing 4 (PIWIL4) followed by quantitative analyses. Moreover, the morphologically defined Adark spermatogonia, which are considered to be the reserve stem cells, were quantified. The PCA and hierarchical clustering revealed three different clusters, one of them containing all Control samples. The main factors driving the sorting of patients to the clusters were the percentage of tubules with elongated spermatids (Cluster 1, all Control patients and two cryptozoospermic patients), the percentage of tubules with spermatocytes (Cluster 2, cryptozoospermic patients), and tubules showing a Sertoli cells only phenotype (Cluster 3, cryptozoospermic patients). Importantly, the percentage of tubules containing elongated spermatids was comparable between Clusters 2 and 3. Additional differences were higher FSH levels (P < 0.001) and lower testicular volumes (P < 0.001) in Cluster 3 compared to Cluster 2. In the spermatogonial compartment of both cryptozoospermic Clusters, we found lower numbers of MAGEA4+ and Adark spermatogonia but higher proportions of PIWIL4+ spermatogonia, which were significantly correlated with a lower percentage of tubules containing elongated spermatids. In line with this common alteration, the outcome of MAR was comparable between Controls as well as both cryptozoospermic Clusters. While we have uncovered the existence of subgroups within the cohort of cryptozoospermic patients, comprehensive genetic analyses remain to be performed to unravel potentially distinct etiologies. The novel insight that cryptozoospermic patients can be divided into two subgroups will facilitate the strategic search for underlying genetic etiologies. Moreover, the shared alterations of the spermatogonial stem cell compartment between the two cryptozoospermic subgroups could represent a general response mechanism to the reduced output of sperm, which may be associated with a progressive phenotype. This study therefore offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in cryptozoospermic patients. German research foundation CRU 326 (grants to: SDP, NN). Moreover, we thank the Faculty of Medicine of the University of Münster for the financial support of Lena Charlotte Schülke through the MedK-program. We acknowledge support from the Open Access Publication Fund of the University of Münster. The authors have no potential conflicts of interest. N/A.

WWC2 expression in the testis: Implications for spermatogenesis and male fertility.

The family of WWC proteins is known to regulate cell proliferation and organ growth control via the Hippo signaling pathway. As WWC proteins share a similar domain structure and a common set of interacting proteins, they are supposed to fulfill compensatory functions in cells and tissues. While all three WWC family members WWC1, WWC2, and WWC3 are found co-expressed in most human organs including lung, brain, kidney, and liver, in the testis only WWC2 displays a relatively high expression. In this study, we investigated the testicular WWC2 expression in spermatogenesis and male fertility. We show that the Wwc2 mRNA expression level in mouse testes is increased during development in parallel with germ cell proliferation and differentiation. The cellular expression of each individual WWC family member was evaluated in published single-cell mRNA datasets of murine and human testes demonstrating a high WWC2 expression predominantly in early spermatocytes. In line with this, immunohistochemistry revealed cytosolic WWC2 protein expression in primary spermatocytes from human testes displaying full spermatogenesis. In accordance with these findings, markedly lower WWC2 expression levels were detected in testicular tissues from mice and men lacking germ cells. Finally, analysis of whole-exome sequencing data of male patients affected by infertility and unexplained severe spermatogenic failure revealed several heterozygous, rare WWC2 gene variants with a proposed damaging function and putative impact on WWC2 protein structure. Taken together, our findings provide novel insights into the testicular expression of WWC2 and show its cell-specific expression in spermatocytes. As rare WWC2 variants were identified in the background of disturbed spermatogenesis, WWC2 may be a novel candidate gene for male infertility.

O-210 Cluster analysis reveals two hidden subgroups among patients with cryptozoospermia

Abstract Study question Can cryptozoospermic patients be sub classified based on clinical parameters? Summary answer Cryptozoospermic patients can be subdivided in two subgroups based on the histological phenotype of their testicular tissues, testicular volume and FSH levels. What is known already Cryptozoospermia is a severe form of oligozoospermia in which patients present with a sperm concentration of ≤ 0.1 million per milliliter. Due to such low sperm concentration, cryptozoospermic patients depend on the surgical procedure of testicular sperm extraction (TESE) to retrieve sperm for intracytoplasmic sperm injection (ICSI). The general etiology underlying cryptozoospermia remains hitherto unknown, likely due to the high heterogeneity within this patient cohort regarding histological and endocrine parameters. Study design, size, duration We retrospectively selected 132 cryptozoospermic patients (Crypto) who underwent TESE during their fertility treatment. Exclusion parameters were genetic diseases, a history of cryptorchidism or tumors and hormonal treatment. As controls, we selected 160 patients with obstructive azoospermia and normal spermatogenesis. All 292 patients were used for principal component analysis (PCA) followed by hierarchical clustering on principal components (HCPC). Participants/materials, setting, methods PCA and hierarchical clustering were performed considering age, testicular volume, hormonal values (FSH, LH, testosterone and free testosterone), ejaculate parameters (Ejaculate pH and volume, sperm concentration and total sperm count) and histological information. Histological analyses were performed on two PAS-stained sections from two independent biopsies per testis and patient. The percentage of seminiferous tubules containing elongated spermatids, round spermatids, spermatocytes or spermatogonia, as well as Sertoli cell only and hyalinized tubules was assessed. Main results and the role of chance The PCA and hierarchical clustering subdivided the patient cohort into 3 different clusters. Cluster 1 included all the controls (n = 160) and 2 Crypto patients. Remaining Crypto patients were equally subdivided between cluster 2 (n = 65) and cluster 3 (n = 65). The control patients in cluster 1 were characterized by the highest percentages of tubules with elongated spermatids (&amp;gt;80%), normal testicular volume and hormonal values. Characteristic of Crypto patients in cluster 2 was the arrest of germ cell differentiation at the level of spermatocytes in the majority of seminiferous tubules. In contrast, the majority of seminiferous tubules in Crypto patients in cluster 3 showed a Sertoli cell only phenotype or even tubular shadows. Interestingly, the more severe histological phenotype of the cluster 3 patients was accompanied by higher FSH and LH levels as well as lower testicular volume compared to cluster 2 patients. Despite this, the percentage of tubules with full spermatogenesis was similar in the two Crypto groups and no difference was found in the sperm retrieval rate after TESE in the two patient groups. Limitations, reasons for caution Uncovering the existence of subgroups within the cohort of Crypto-patients is the prerequisite for unveiling potentially distinct etiologies. Comprehensive genetic and scRNA-seq analyses of these Crypto-subgroups remain to be performed to validate the findings. Wider implications of the findings Overall, this study offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in Crypto patients and the division into two subgroups will facilitate the strategic search for underlying causes. Moreover, this approach could be applied to any infertility type in order to identify hidden subgroups. Trial registration number not applicable

Lack of germline transmission in male mice following a single intravenous administration of AAV5-hFVIII-SQ gene therapy

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype five gene therapy under investigation for the treatment of hemophilia A. Herein, we assessed the potential for germline transmission of AAV5-hFVIII-SQ in mice. Male B6.129S6-Rag2tm1Fwa N12 mice received a single intravenous dose of vehicle or 6 × 1013 vg/kg AAV5-hFVIII-SQ. Vehicle and AAV5-hFVIII-SQ-treated mice were mated with naïve females 4 days after dosing, when the concentration of vector genomes was expected to be at its peak in semen, and 37 days after dosing, when a full spermatogenesis cycle was estimated to be complete. Quantitative PCR was used to evaluate the presence of transgene DNA in liver and testes from F0 males dosed with AAV5-hFVIII-SQ and liver tissue of F1 offspring. Transgene DNA was detected in liver and testes of all F0 males dosed with AAV5-hFVIII-SQ, confirming successful transduction. Importantly, no transgene DNA was detected in any tested F1 offspring derived from F0 males dosed with AAV5-hFVIII-SQ. Using a novel 2-stage statistical model that takes into account the number of males dosed with AAV5-hFVIII-SQ and the number of offspring sired by these males, we estimate that the risk of germline transmission is <5% with a 99.2% confidence level.

Histological study on the influence of puberty suppression and hormonal treatment on developing germ cells in transgender women.

STUDY QUESTIONCan transgender women cryopreserve germ cells obtained from their orchiectomy specimen for fertility preservation, after having used puberty suppression and/or hormonal treatment?SUMMARY ANSWERIn the vast majority of transgender women, there were still immature germ cells present in the orchiectomy specimen, and in 4.7% of transgender women—who all initiated medical treatment in Tanner stage 4 or higher—mature spermatozoa were found, which would enable cryopreservation of spermatozoa or testicular tissue after having used puberty suppression and/or hormonal treatment.WHAT IS KNOWN ALREADYGender affirming treatment (i.e. puberty suppression, hormonal treatment, and subsequent orchiectomy) impairs reproductive function in transgender women. Although semen cryopreservation is generally offered during the transition process, this option is not feasible for all transgender women (e.g. due to incomplete spermatogenesis when initiating treatment in early puberty, in case of inability to masturbate, or when temporary cessation of hormonal treatment is too disruptive). Harvesting mature spermatozoa, or testicular tissue harboring immature germ cells, from orchiectomy specimens obtained during genital gender-affirming surgery (gGAS) might give this group a chance of having biological children later in life. Previous studies on spermatogenesis in orchiectomy specimens showed conflicting results, ranging from complete absence of germ cells to full spermatogenesis, and did not involve transgender women who initiated medical treatment in early- or late puberty.STUDY DESIGN, SIZE, DURATIONHistological and immunohistochemical analyses were performed on orchiectomy specimens from 214 transgender women who underwent gGAS between 2006 and 2018. Six subgroups were identified, depending on pubertal stage at initiation of medical treatment (Tanner stage 2-3, Tanner stage 4-5, adult), and whether hormonal treatment was continued or temporarily stopped prior to gGAS in each of these groups.PARTICIPANTS/MATERIALS, SETTING, METHODSAll transgender women used a combination of estrogens and testosterone suppressing therapy. Orchiectomy specimen sections were stained with Mayer’s hematoxylin and eosin and histologically analyzed to assess the Johnsen score and the ratio of most advanced germ cell types in at least 50 seminiferous tubular cross-sections. Subsequently, immunohistochemistry was used to validate these findings using spermatogonia, spermatocytes or spermatids markers (MAGE-A3/A4, γH2AX, Acrosin, respectively). Possibilities for fertility preservation were defined as: preservation of spermatozoa, preservation of spermatogonial stem cells or no possibilities (in case no germ cells were found). Outcomes were compared between subgroups and logistic regression analyses were used to assess the association between the duration of hormonal treatment and the possibilities for fertility preservation.MAIN RESULTS AND THE ROLE OF CHANCEMature spermatozoa were encountered in 4.7% of orchiectomy specimens, all from transgender women who had initiated medical treatment in Tanner stage 4 or higher. In 88.3% of the study sample orchiectomy specimens only contained immature germ cells (round spermatids, spermatocytes or spermatogonia, as most advanced germ cell type). In 7.0%, a complete absence of germ cells was observed, all these samples were from transgender women who had initiated medical treatment in adulthood. Cessation of hormonal treatment prior to gGAS did not affect the presence of germ cells or their maturation stage, nor was there an effect of the duration of hormonal treatment prior to gGAS.LIMITATIONS, REASONS FOR CAUTIONSince data on serum hormone levels on the day of gGAS were not available, we were unable to verify if the transgender women who were asked to temporarily stop hormonal treatment 4 weeks prior to surgery actually did so, and if people with full spermatogenesis were compliant to treatment.WIDER IMPLICATIONS OF THE FINDINGSThere may still be options for fertility preservation in orchiectomy specimens obtained during gGAS since a small percentage of transgender women had full spermatogenesis, which could enable cryopreservation of mature spermatozoa via a testicular sperm extraction procedure. Furthermore, the vast majority still had immature germ cells, which could enable cryopreservation of testicular tissue harboring spermatogonial stem cells. If maturation techniques like in vitro spermatogenesis become available in the future, harvesting germ cells from orchiectomy specimens might be a promising option for those who are otherwise unable to have biological children.STUDY FUNDING/COMPETING INTERESTNone.TRIAL REGISTRATION NUMBERN/A.

Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1−/− mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1−/− mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1−/− compared to wild-type mice. Furthermore, cultured Uch-l1−/− SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

Chemotherapy induced damage to spermatogonial stem cells in prepubertal mouse in vitro impairs long-term spermatogenesis.

Chemotherapy can affect testis development of young boys with cancer, reducing the chances of fatherhood in adulthood. Studies using experimental models are needed to determine the damage caused by individual chemotherapy drugs in order to predict the risk of infertility and direct patients towards appropriate fertility preservation options. Here, we investigated the individual role of two drugs, cisplatin and doxorubicin, using an in vitro culture model of prepubertal (postnatal day 5) mouse testis that supports induction and maintenance of full spermatogenesis. Twenty-four hour exposure with either drug at clinically-relevant doses (0.25, 0.5 or 0.75 μg/mL for cisplatin, or 0.01, 0.03 or 0.05 μg/mL for doxorubicin), induced an acute significant loss of spermatogonial stem cells (SSCs; PLZF+), proliferating SSCs (PLZF+BrdU+), total germ cells (MVH+), and spermatocytes (SCP3+) one week after chemotherapy exposure. By the time of the first (Week 4) and second (Week 8) waves of spermatogenesis, there was no longer any effect on SSC or proliferating SSC numbers in drug-exposed testis compared to untreated tissue: however, the populations of total germ cells and spermatocytes were still lower in the higher-dose cisplatin treated groups, along with a reduced frequency of round and elongated spermatids in both cisplatin- and doxorubicin-treated testis fragments. Overall, this study details a direct impairment of germ cell development following acute chemotherapy-induced damage during the prepubertal phase, most likely due to an effect on SSCs, using an in vitro culture system that successfully recapitulates key events of mouse spermatogenesis.

Characterisation of testicular function and spermatogenesis in transgender women.

Does gender-affirming treatment prevent full spermatogenesis in transgender women (TW)? Adequate hormonal therapy (HT) leads to complete suppression of spermatogenesis in most TW, if serum testosterone levels within female reference ranges are obtained. Gender-affirming treatment in transgender individuals may involve gender-affirming HT. The effects on spermatogenesis in TW remain unclear. In order to add information from a referral centre for transgender care, we wish to compare results of earlier studies with our population of TW who received a standard hormone treatment. This was a prospective cohort study part of the European Network for the Investigation of Gender Incongruence (ENIGI), conducted between 15 February 2010 and 30 September 2015. There were 162 TW were included in the ENIGI study at the Ghent University Hospital in Belgium. Participants are included in ENIGI when they first start HT, and follow-up visits occur over the next 3 years. The study included 97 TW who initiated HT with cyproterone acetate (CPA) plus oestrogens and proceeded with gonadectomy at the Ghent University Hospital. Testicular tissue retrieved during gonadectomy was processed and stained for four different germ cell markers by the Biology of the Testis lab at the Vrije Universiteit Brussel. Subsequent immunohistochemical staining was performed for melanoma-associated antigen A4 (MAGE-A4, marker for spermatogonia and early spermatocytes), boule homologue, RNA-binding protein (BOLL, marker for secondary spermatocytes and round spermatids), cAMP-responsive element modulator (CREM, marker for round spermatids) and acrosin (marker for acrosome visualization). Serum levels of sex steroids were measured prior to surgery. Suppressed testosterone levels (<50 ng/dl) were found in 92% of the participants prior to surgery. The mean time between initiation of HT and surgery was 685 days. In 88% (85/97) of the sections, MAGE-A4 staining was positive. Further staining could not reveal complete spermatogenesis in any participant. Testicular function of the participants prior to initiation of HT was not assessed, although all participants presented with cisgender male serum testosterone values before initiation of HT. The current study only reports on people using CPA at a fixed dose and may therefore not be applicable to all TW. HT leads to complete suppression of spermatogenesis in most TW, if serum testosterone levels within female reference ranges are obtained. Serum testosterone levels are associated with the sperm maturation rate. It is important to discuss sperm preservation before the start of hormone therapy. If serum testosterone levels remain higher, spermatogenesis may still occur. D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2819N). Processing of the testis specimens was funded by the Biology of The Testes (BITE) research group (Department of Reproduction, Genetics and Regenerative medicine at Vrije Universiteit Brussel (VUB)). There are no competing interests. N/A.

Shifts in Reproductive Investment in Response to Competitors Lower Male Reproductive Success.

AbstractIn many species, males exhibit phenotypic plasticity in sexually selected traits when exposed to social cues about the intensity of sexual competition. To date, however, few studies have tested how this plasticity affects male reproductive success. We initially tested whether male mosquitofish, Gambusia holbrooki (Poeciliidae), change their investment in traits under pre- and postcopulatory sexual selection depending on the social environment. For a full spermatogenesis cycle, focal males were exposed to visual and chemical cues of rivals that were either present (competitive treatment) or absent (control). Males from the competitive treatment had significantly slower-swimming sperm but did not differ in sperm count from control males. When two males competed for a female, competitive treatment males also made significantly fewer copulation attempts and courtship displays than control males. Further, paternity analysis of 708 offspring from 148 potential sires, testing whether these changes in reproductive traits affected male reproductive success, showed that males previously exposed to cues about the presence of rivals sired significantly fewer offspring when competing with a control male. We discuss several possible explanations for these unusual findings.

Impact of a nutritional supplement (Impryl) on male fertility: study protocol of a multicentre, randomised, double-blind, placebo-controlled clinical trial (SUppleMent Male fERtility, SUMMER trial)

IntroductionInfertility is a worldwide problem and about 10%–15% of all couples will be affected by the inability to have children. In approximately 50% of infertile couples, a male factor is...

Exposure to Bisphenol A affect the size, viability, and maturation of pig testicular tissue ectopically grafted in immunocompromised mice

Bisphenol A (BPA) is a plastic additive, specially of polycarbonates and epoxy resins, as it imparts transparency, flexibility, and durability. BPA‐containing plastic is present in a great variety of common consumer products, such as water bottles, food packaging, and coating of food cans. BPA leaches from these products into the environment and is detectable in humans, animals, and drinking water sources. In rodents, BPA exhibits estrogen‐like properties and has detrimental effects on testicular maturation and function. Currently the effects of BPA in other species are still unclear. However, the studies that are necessary to elucidate the effects of potentially toxic compounds on the testes of non‐rodent species are logistically difficult and ethically prohibitive. One alternative is using mice as bioincubators to grow testicular tissue of large animals and then expose these mice to those compounds. In this technique, small pieces of immature testes transplanted from these animals into adult castrated immunocompromised mice, survive and fully mature. In order to investigate the effects of BPA on the testes of other species, we grafted piglet testicular pieces (~1 mg, 8 pieces / mouse) under the dorsal skin of adult castrated NCr nude mice. After two weeks of grafting, mice (n=10) were treated daily by oral gavage for 26 weeks (the approximate time in which pig testicular tissue reaches full maturity in mice) with 10 mg/kg of BPA in corn oil as vehicle. As control, we exposed an equal number of mice to the same volume of corn oil. The experimental dose of BPA is lower than the lowest adverse effect level (LOAEL, 50 mg/kg) for humans, which is the currently accepted by the US Environmental Protection Agency. At sacrifice, neither the weight of mice nor their liver were affected by treatment (P &gt; 0.05), indicating that subsequent effects of treatment on the grafted tissue were a result of direct BPA exposure and not due to indirect systemic toxicity induced by this compound. The rate of graft recovery was not affected by treatment either; 65.62 vs 58.33 % of the transplanted pieces were recovered from the treated and control mice, respectively (P&gt;0.05). However, the graft weight was significantly reduced by BPA; 24.56 ± 4.33 vs 43.62 ± 5.36 mg (P&lt;0.05). Moreover, the rate of graft viability (defined as the presence of viable seminiferous tubules in the graft) was significantly reduced in treated mice as well (30.43 vs 70.00 % in treated and control groups, respectively, P&lt;0.05). Some seminiferous tubules in grafts of both groups of mice reached full maturity (full spermatogenesis; presence of elongated spermatids in the seminiferous epithelium), but the number of these tubules was lower in the BPA‐treated group; 2.43 vs 8.89 % (P&lt;0.05). In summary, exposure to BPA at these low levels results in reduced size, viability, and maturation of the piglet testicular tissue ectopically grafted in mice. This strongly suggests that the testes of non‐rodent animals are also susceptible to the detrimental effects observed in rodents with exposure to BPA.Support or Funding InformationMidwestern University intramural funds

Dibutyl phthalate affects the recovery, size, and viability of pig testicular tissue ectopically grafted in immunocompromised mice

Phthalic acid esters, or phthalates, are used as plasticizers and are present in polyvinyl chloride products, solvents, lubricating oils, and personal care products. Phthalates are not covalently bound to these products and easily leach into the environment, resulting in ubiquitous exposure of humans and animals. Phthalates have been shown to have anti‐androgenic effects in rodents, which makes it an important disruptor of testicular maturation and function. Some studies have been performed in larger mammals, including primates; however, the results are still inconclusive. The studies that are necessary to elucidate the effects of phthalates on the testes of non‐rodent species are logistically difficult and ethically prohibitive. One alternative to assess the effects of these compounds on the testes of non‐rodent species is using mice as bioincubators to grow testicular tissue of large animals and then expose these mice to those compounds. In this technique, small pieces of immature testes transplanted from large animals into adult castrated immunocompromised mice, survive and fully mature. In order to investigate the effects of this compound on the testes of other species, we grafted piglet testicular pieces (~1 mg, 8 pieces / mice) under the skin of adult castrated NCr nude mice. After two weeks of grafting, mice (n=10) were treated daily by oral gavage for 26 weeks (the approximate time in which pig testicular tissue reaches full maturity in mice) with 10 mg/kg of Dibutyl phthalate (DBP) in corn oil as vehicle. As control, we exposed an equal number of mice to the same volume of corn oil. The experimental dose of DBP used is equivalent to dose exposures that may be encountered by humans (for example, children exposed to medical supplies in the intensive care unit). At sacrifice, neither the weight of mice nor their liver were affected by treatment (P&gt;0.05), indicating that effects of treatment on the grafted tissue were a result of direct DBP exposure and not due to indirect systemic toxicity induced by this compound. The rate of graft recovery was affected by treatment; 26.78 vs 58.33 % of the transplanted pieces were recovered from the treated and control mice, respectively (P&lt;0.05). The graft weight was also significantly reduced by DBP; 17.16 ± 4.33 vs 43.62 ± 5.36 mg (P&lt;0.05). Moreover, the rate of graft viability (defined as the presence of viable seminiferous tubules in the graft) was significantly reduced in treated mice as well (30.00 vs 70.00 % in treated and control groups, respectively, P&lt;0.05). Some seminiferous tubules in grafts of both groups of mice reached full maturity (full spermatogenesis; presence of elongated spermatids in the seminiferous epithelium), the number of these tubules was not affected by DBP; 6.41 vs 8.89 % (P&gt;0.05). In summary, exposure to DBP at a dose that mimics possible environmental exposures, reduced the recovery, size, and viability of the piglet testicular tissue ectopically grafted into the mice, but not its maturity. Future studies will be aimed to determine the effects of DBP on the function of the grafts (e.g., steroidogenesis). These results suggests that the testes of non‐rodent animals are also susceptible to the detrimental effects observed in rodents after expose to DBP.Support or Funding InformationMidwestern University intramural funds

Evaluating genetic causes of azoospermia: What can we learn from a complex cellular structure and single-cell transcriptomics of the human testis?

Azoospermia is a condition defined as the absence of spermatozoa in the ejaculate, but the testicular phenotype of men with azoospermia may be very variable, ranging from full spermatogenesis, through arrested maturation of germ cells at different stages, to completely degenerated tissue with ghost tubules. Hence, information regarding the cell-type-specific expression patterns is needed to prioritise potential pathogenic variants that contribute to the pathogenesis of azoospermia. Thanks to technological advances within next-generation sequencing, it is now possible to obtain detailed cell-type-specific expression patterns in the testis by single-cell RNA sequencing. However, to interpret single-cell RNA sequencing data properly, substantial knowledge of the highly sophisticated data processing and visualisation methods is needed. Here we review the complex cellular structure of the human testis in different types of azoospermia and outline how known genetic alterations affect the pathology of the testis. We combined the currently available single-cell RNA sequencing datasets originating from the human testis into one dataset covering 62,751 testicular cells, each with a median of 2637 transcripts quantified. We show what effects the most common data-processing steps have, and how different visualisation methods can be used. Furthermore, we calculated expression patterns in pseudotime, and show how splicing rates can be used to determine the velocity of differentiation during spermatogenesis. With the combined dataset we show expression patterns and network analysis of genes known to be involved in the pathogenesis of azoospermia. Finally, we provide the combined dataset as an interactive online resource where expression of genes and different visualisation methods can be explored ( https://testis.cells.ucsc.edu/ ).

Establishment of Etv5 gene knockout mice as a recipient model for spermatogonial stem cell transplantation.

ABSTRACTSpermatogonial stem cell (SSC) transplantation is an alternative reproductive method to achieve conservation and production of elite animals in livestock production. Creating a recipient animal without endogenous germ cells is important for effective SSC transplantation. However, natural mutants with depletion of SSCs are difficult to obtain, and drug ablation of endogenous germ cells is arduous to perform for practical use. In this study, we used mouse models to study the preparation of recipients with congenital germ cell ablation. We knocked out (KO) Ets-variant gene 5 (Etv5) in mice using the CRISPR/Cas9 system. The testicular weight of Etv5−/− mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in Etv5−/− mice. At 12 weeks of age, the tubules of Etv5−/− mice lacked almost all spermatogenic cells with a Sertoli cell-only phenotype, and sperm were completely absent in the epididymis. We subsequently transplanted allogeneic SSCs with enhanced green fluorescent protein (EGFP) into 3- (immature) or 7-week-old (mature) Etv5−/− mice. Partially restoration of germ cell layers in the seminiferous tubules and spermatogenesis was observed in all immature testes but not in mature adult testes at 2 months post-transplantation. The presence of heterologous genes Etv5 and EGFP in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although Etv5−/− mice could accommodate and support foreign germ cell transplantation, this process occurs in a quite low efficiency to support a full spermatogenesis of transplanted SSCs. However, using Etv5−/− mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.

Rebuilding the human testis in vitro

Increasing rates of male infertility have led to a greater need for relevant model systems to gain further insight into male fertility and its failings. Spermatogenesis and hormone production occur within distinct regions of the testis. Defined by specialized architecture and a diverse population of cell types, it is no surprise that disruption of this highly organized microenvironment can lead to infertility. To date, no robust in vitro system has facilitated full spermatogenesis resulting in the production of fertilization‐competent human spermatozoa. Here, we review a selection of current in vitro systems available for modelling the human testis microenvironment with focus on the progression of spermatogenesis and recapitulation of the testis microenvironment.

RNA profiling of human testicular cells identifies syntenic lncRNAs associated with spermatogenesis.

Is the noncoding transcriptional landscape during spermatogenesis conserved between human and rodents? We identified a core group of 113 long noncoding RNAs (lncRNAs) and 20 novel genes dynamically and syntenically transcribed during spermatogenesis. Spermatogenesis is a complex differentiation process driven by a tightly regulated and highly specific gene expression program. Recently, several studies in various species have established that a large proportion of known lncRNAs are preferentially expressed during meiosis and spermiogenesis in a testis-specific manner. To further investigate lncRNA expression in human spermatogenesis, we carried out a cross-species RNA profiling study using isolated testicular cells. Human testes were obtained from post-mortem donors (N = 8, 51 years old on average) or from prostate cancer patients with no hormonal treatment (N = 9, 80 years old on average) and only patients with full spermatogenesis were used to prepare enriched populations of spermatocytes, spermatids, Leydig cells, peritubular cells and Sertoli cells. To minimize potential biases linked to inter-patient variations, RNAs from two or three donors were pooled prior to RNA-sequencing (paired-end, strand-specific). Resulting reads were mapped to the human genome, allowing for assembly and quantification of corresponding transcripts. Our RNA-sequencing analysis of pools of isolated human testicular cells enabled us to reconstruct over 25 000 transcripts. Among them we identified thousands of lncRNAs, as well as many previously unidentified genes (novel unannotated transcripts) that share many properties of lncRNAs. Of note is that although noncoding genes showed much lower synteny than protein-coding ones, a significant fraction of syntenic lncRNAs displayed conserved expression during spermatogenesis. Raw data files (fastq) and a searchable table (.xlss) containing information on genomic features and expression data for all refined transcripts have been submitted to the NCBI Gene Expression Omnibus under accession number GSE74896. Isolation procedures may alter the physiological state of testicular cells, especially for somatic cells, leading to substantial changes at the transcriptome level. We therefore cross-validated our findings with three previously published transcriptomic analyses of human spermatogenesis. Despite the use of stringent filtration criteria, i.e. expression cut-off of at least three fragments per kilobase of exon model per million reads mapped, fold-change of at least three and false discovery rate adjusted P-values of less than <1%, the possibility of assembly artifacts and false-positive transcripts cannot be fully ruled out. For the first time, this study has led to the identification of a large number of conserved germline-associated lncRNAs that are potentially important for spermatogenesis and sexual reproduction. In addition to further substantiating the basis of the human testicular physiology, our study provides new candidate genes for male infertility of genetic origin. This is likely to be relevant for identifying interesting diagnostic and prognostic biomarkers and also potential novel therapeutic targets for male contraception. This work was supported by l'Institut national de la santé et de la recherche médicale (Inserm); l'Université de Rennes 1; l'Ecole des hautes études en santé publique (EHESP); INERIS-STORM to B.J. [N 10028NN]; Rennes Métropole 'Défis scientifiques émergents' to F.C (2011) and A.D.R (2013). The authors have no competing financial interests.

Human Testis Phosphoproteome Reveals Kinases as Potential Targets in Spermatogenesis and Testicular Cancer

Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular, functional and structural changes. It requires tight regulation, because disturbances in any of the spermatogenic processes would lead to fertility deficiencies as well as disorders in offspring. To increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human male gonad. Metal oxide affinity chromatography using TiO2 combined with LC-MS/MS was conducted to profile the phosphoproteome of adult human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active human protein kinases in the testis were predicted both by the number of phosphopeptide spectra identified and the phosphorylation status of the kinase activation loop. The potential function of cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4 (PAK4) has been explored by in silico, protein-protein interaction analysis, immunodetection in testicular tissue, and a functional assay in a human embryonal carcinoma cell line. The colocalization of CDK12 with Golgi markers suggests a potential crucial role of this protein kinase during sperm formation. PAK4 has been found expressed in human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our protein discovery analysis confirms that phosphoregulation by protein kinases is highly active in sperm differentiation and opens a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility and tumor behavior.

Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate.

STUDY QUESTIONCan full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously?SUMMARY ANSWERIntratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months.WHAT IS KNOWN ALREADYAuto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful.STUDY DESIGN, SIZE, DURATIONIn this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8–1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4–6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation.PARTICIPANTS/MATERIALS, SETTING, METHODSTransplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids.MAIN RESULTS AND THE ROLE OF CHANCEFour months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONAlthough xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants.WIDER IMPLICATIONS OF THE FINDINGSIn this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood.STUDY FUNDING/COMPETING INTEREST(S)This project was funded by the ITN Marie Curie Programme ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

The Sertoli cell: what can we learn from different vertebrate models?

Besides having medical applications, comparative studies on reproductive biology are very useful, providing, for instance, essential knowledge for basic, conservation and biotechnological research. In order to maintain the reproductive potential and the survival of all vertebrate species, both sperm and steroid production need to occur inside the testis. From the approximately fifty thousand vertebrate species still alive, very few species are already investigated; however, our knowledge regarding Sertoli cell biology is quite good. In this regard, it is already known that since testis differentiation the Sertoli cells are the somatic cells in charge of supporting and orchestrating germ cells during development and full spermatogenesis in adult animals. In the present review, we highlight key aspects related to Sertoli cell biology in vertebrates and show that this key testis somatic cell presents huge and intrinsic plasticity, particularly when cystic (fish and amphibians) and non-cystic (reptiles, birds and mammals) spermatogenesis is compared. In particular, we briefly discuss the main aspects related to Sertoli cells functions, interactions with germ cells, Sertoli cells proliferation and efficiency, as well as those regarding spermatogonial stem cell niche regulation, which are crucial aspects responsible for the magnitude of sperm production. Most importantly, we show that we could greatly benefit from investigations using different vertebrate experimental models, mainly now that there is a big concern regarding the decline in human sperm counts caused by a multitude of factors.