Number Of Germ Cells Research Articles

Identification and Manipulation of Spermatogonial Stem Cells with the Aim of Inducing Spermatogenesis in Vitro.

Assisted reproduction techniques for infertile men with non-obstructive azoospermia require a sufficient number of functional germ cells produced in vitro. Understanding the mechanisms that allow the resumption of spermatogenesis outside the testicular environment is crucial for fertility preservation in these patients. A review of the literature was conducted using databases such as PubMed, Scopus and Web of Science, with keywords including "spermatogonial stem cell," "germ cells," "male factor infertility," and "enrichment and propagation of SSCs in vitro." Currently, two models-"in vivo" and "in vitro"-have been developed for producing haploid germ cells. The "in vivo" models include spermatogonial stem cell transplantation and testicular xenograft techniques. In contrast, the "in vitro" models consist of conventional culture systems, organ culture, and three-dimensional culture systems, all designed to induce spermatogenesis in vitro. These culture systems enable the simulation of the seminiferous epithelium in vitro, which facilitates better regulation of cell-signaling pathways that control the self-renewal and differentiation of SSCs. This review provides up-to-date information on the organization of SSCs, focusing on the identification, proliferation, and differentiation of spermatogonia in vitro.

Near-stop in spermatogonia progression and reduced sperm motility in two testis forms of captive North African catfish in Kalasin, Thailand

North African catfish (Clarias gariepinus) is integral to aquaculture, particularly in hybrid production with bighead catfish (Clarias macrocephalus). Recent declines in their production output have been linked to the quality of the parental sperm. This study aimed to evaluate the state of spermatogenesis in two morphological testis forms, normal white testes (CGA_A) and black-brown testes (CGA_B), in captive populations to understand their differences in the cytological processes of spermatogenesis and impact on sperm quality and fertility. We performed computer-assisted sperm analysis (CASA), meiotic chromosome analysis, gene expression profiling, histological examination, and identification of apoptotic cells using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Additionally, microsatellite genotyping was conducted to assess genetic diversity of the populations, showing that similar gene pool patterns were observed between the two populations with the CGA_A and the CGA_B testes. The average sperm motility was 11.15 %. Histologically, the CGA_A testes predominantly contained spermatozoa. By contrast, the CGA_B testes, characterized by black-brown color and harder texture, were composed mainly of spermatocytes and exhibited a higher incidence of apoptotic spermatogonia. Additionally, the CGA_B testes displayed reduced expression of genes essential for normal testicular development. Notably, the CGA_A testes showed significantly higher expression of GtH I, GtH II, Dazl, and Cxcr4b genes and lower levels of Caspase-3 and p53 (p < 0.05). The selective removal of deficient spermatogonia through early apoptosis in CGA_B testes suggest a mechanism contributing to male infertility due to imbalances between the number of germ cells and Sertoli cells. Despite the use of CGA_A testis for fertilization, a hatching rate in the captive populations was 36.43 % over the last three years (2021−2023), substantially lower than that previously reported, indicating the presence of genetic and environmental factors that influence reproductive success in North African catfish. Differences in spermatogenesis between testis forms highlight the need for genetic and environmental strategies to enhance reproductive outcomes in aquaculture.

Maternal dnd1 is essential for migration and maintenance of PGCs in Nile tilapia at larval stage

As a germ-cell-specific maternal mRNA and RNA binding protein, dead end (dnd1) plays an important role in migration and maintenance of primordial germ cells (PGCs), and therefore, disruption of dnd1 results in no germ cells. However, the effect and duration of maternal dnd1 on PGCs in zygote mutants have not been reported yet. In the present study, CRISPR/Cas9 was used to mutate dnd1 in tilapia. Supported by the maternal dnd1 mRNA, germ cells of the dnd1 homozygous mutants proliferated and migrated normally before 7 dpf (days post fertilization). After the degradation of maternal dnd1 mRNA, significant downregulation of piwil1, piwil2, nanos2, nanos3 and vasa mRNA, and blocked migration of germ cells were observed at 14 dpf. In dnd1 zygote mutants, the number of germ cells was decreased significantly compared with wild type (WT) at 25 dpf, and no germ cell was observed at 30 dpf. Consistently, no expression of vasa and nanos3 was detected in the adult dnd1 mutants by qPCR. Interestingly, Cyp19a1a/cyp19a1a and foxl2 were expressed in dnd1-deficient XX gonads, while Cyp11c1/cyp11c1 and dmrt1 were expressed in dnd1-deficient XY gonads as in WT gonads. Correspondingly, both serum E2 level in XX and 11-KT level in XY fish did not differ significantly between dnd1 mutant and WT fish. Taken together, the present study demonstrates that disruption of dnd1 lead to progressive loss of germ cells, while the somatic cells are not affected in tilapia. The sexual fate of gonads is determined by the genetic sex instead of germ cell number, which makes tilapia a potential surrogate model for germ cell transplantation.

Medaka (Oryzias latipes) Dmrt3a Is Involved in Male Fertility.

Research across various species has demonstrated that the doublesex and mab-3-related transcription factor 3 (dmrt3) plays pivotal roles in testis development. However, the precise molecular mechanisms of dmrt3 remain unclear. In this study, we investigated the role of dmrt3 (dmrt3a) in testis development using the model organism medaka (Oryzias latipes). SqRT-PCR and ISH analyses revealed that dmrt3a is predominantly expressed in the testis, especially in the spermatid and spermatozoon. Using CRISPR/Cas9, we generated two dmrt3a homozygous mutants (-8 bp and -11 bp), which exhibited significantly reduced fertilization rates and embryo production. Additionally, the number of germ cells and sperm motility were markedly decreased in the dmrt3a mutants, manifesting as the symptoms of asthenozoospermia and oligozoospermia. Interestingly, RNA-Seq analysis showed that the deficiency of dmrt3a could lead to a significant downregulation of numerous genes related to gonadal development and severe disruptions in mitochondrial function. These results suggested that dmrt3a is essential for spermatogenesis and spermatozoa energy production. This paper provides new insights and perspectives for further exploring the molecular mechanisms underlying spermatogenesis and addressing male reproductive issues.

Spermatogenesis in mouse testicular organoids with testis-specific architecture, improved germ cell survival and testosterone production

This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.

Diverse somatic Transformer and sex chromosome karyotype pathways regulate gene expression in Drosophila gonad development.

The somatic sex determination gene transformer (tra) is required for the highly sexually dimorphic development of most somatic cells, including those of the gonads. In addition, somatic tra is required for the germline development even though it is not required for sex determination within germ cells. Germ cell autonomous gene expression is also necessary for their sex determination. To understand the interplay between these signals, we compared the phenotype and gene expression of larval wild-type gonads and the sex-transformed tra gonads. XX larval ovaries transformed into testes were dramatically smaller than wild-type, with significant reductions in germ cell number, likely due to altered geometry of the stem cell niche. Additionally, there was a defect in progression into spermatocyte stages. XY larval testes transformed into ovaries had excessive germ cells, possibly due to the earlier onset of cell division. We suggest that germ cells are neither fully female nor male following somatic sex transformation, with certain pathways characteristic of each sex expressed in tra mutants. We found multiple patterns of somatic and germline gene expression control exclusively due to tra, exclusively due to sex chromosome karyotype, but usually due to a combination of these factors showing tra and sex chromosome karyotype pathways regulate gene expression during Drosophila gonad development.

Depletion of placental brain-derived neurotrophic factor (BDNF) is attributed to premature ovarian insufficiency (POI) in mice offspring

IntroductionPremature ovarian insufficiency (POI) is one of the causes of female infertility. Unexplained POI is increasingly affecting women in their reproductive years. However, the etiology of POI is diverse and remains elusive. We and others have shown that brain-derived neurotrophic factor (BDNF) plays an important role in adult ovarian function. Here, we report on a novel role of BDNF in the Developmental Origins of POI.MethodsPlacental BDNF knockout mice were created using CRISPR/CAS9. Homozygous knockout (cKO(HO)) mice didn’t survive, while heterozygous knockout (cKO(HE)) mice did. BDNF reduction in cKO(HE) mice was confirmed via immunohistochemistry and Western blots. Ovaries were collected from cKO(HE) mice at various ages, analyzing ovarian metrics, FSH expression, and litter sizes. In one-month-old mice, oocyte numbers were assessed using super-ovulation, and oocyte gene expression was analyzed with smart RNAseq. Ovaries of P7 mice were studied with SEM, and gene expression was confirmed with RT-qPCR. Alkaline phosphatase staining at E11.5 and immunofluorescence for cyclinD1 assessed germ cell number and cell proliferation.ResultscKO(HE) mice had decreased ovarian function and litter size in adulthood. They were insensitive to ovulation induction drugs manifested by lower oocyte release after superovulation in one-month-old cKO(HE) mice. The transcriptome and SEM results indicate that mitochondria-mediated cell death or aging might occur in cKO(HE) ovaries. Decreased placental BDNF led to diminished primordial germ cell proliferation at E11.5 and ovarian reserve which may underlie POI in adulthood.ConclusionThe current results showed decreased placental BDNF diminished primordial germ cell proliferation in female fetuses during pregnancy and POI in adulthood. Our findings can provide insights into understanding the underlying mechanisms of POI.

P-069 The expression patterns of ALOX15 and GPX4 in the seminiferous epithelium of men with non-obstructive azoospermia (NOA) due to primary testicular failure

Abstract Study question Does the expression of ALOX15 and GPX4 differ in NOA men with a history of cryptozoospermia from NOA who never had sperm in their ejaculate? Summary answer There was no significant difference between these 2 groups of patients. What is known already Oxidative stress (OS) has been investigated as a prime suspect for many unexplained pathologies, including primary testicular failure (PTF). The fact that male germ cells are rich in polyunsaturated fatty acids (PUFAs) but have a limited amount of cytoplasmic scavenging enzymes, is the reason behind their extreme vulnerability to OS. The accumulation of lipid peroxidation markers leads to the inactivation of glutathione peroxidase 4 (GPX4), the lipid repair enzyme, and the exacerbation of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that promotes lipid degradation. This study investigates the activity of these antagonizing enzymes in the testicular tissue of NOA patients. Study design, size, duration This observational study was conducted in the period from May 2020, following the Ethics Committee of the University of Split School of Medicine approval, to October 2023. The testicular tissue from 57 patients diagnosed with NOA (according to WHO criteria) and referred to their first microTESE as a part of fertility treatment was collected and processed. Participants/materials, setting, methods Samples were prepared for histological evaluation and immunodetection (fixation; embedding in paraffin; H&amp;E staining; and staining with anti-ALOX15 and anti-GPX4 antibodies). Two groups of NOA patients were observed: Crypto-NOA and NOA group. The main outcome measure was the number of ALOX15 positive germ cells per 1 mm² of the seminiferous tubule surface (N/ mm²) and the percentage (%) of GPX4 positive surface per surface of the tubule measured by the ImageJ program. Main results and the role of chance Spermatozoa were retrieved in 37 of 57 (64,91 %) men; samples of 4 patients had to be excluded due to poor fixation. There were 18 patients in the Crypto-NOA group, and 10 in the NOA group, while 5 patients were diagnosed with obstructive azoospermia (OA) and were used as controls. For comparison of the baseline data of Crypto-NOA and NOA patients, we used the Mann-Whitney test, and with statistical significance set at the level of P &amp;lt; 0.05, we found that there were significantly more patients with cryptorchism in the NOA group. As a tool for histological evaluation, we used the diagnostic code designed by McLachlan et al. and found similar spermatogenic phenotypes between Crypto NOA and NOA patients. The most represented phenotype was hypospermatogenesis with a moderate reduction in late spermatid number mixed with normal tubules (38,8 % in Crypto-NOA; 40% in NOA), following the phenotype where late spermatids are present in some tubules but mixed with tubules showing no progression past the spermatocyte stage. Using the Mann-Whitney test, we did not find a significant difference in the expression of ALOX15 and GPX4 in the seminiferous epithelium of Crypto-NOA and NOA patients. Limitations, reasons for caution The testicular tissue of NOA patients is characterized by focal or patchy spermatogenesis which led to the exclusion of several samples due to the fixation of tissue with a homogenous pattern of Sertoli cell-only or germ-cell arrest. Wider implications of the findings This study will provide additional information regarding the cause of impaired spermatogenesis and help to determine if the patient is a potential candidate for antioxidant therapy. Our next step is to associate the expression patterns of ALOX15 and GPX4 with the success rates of in vitro fertilization procedure outcomes. Trial registration number not applicable

Tebuconazole Induces Mouse Fetal Testes Damage via ROS Generation in an Organ Culture Method.

The fungicide tebuconazole (TEB) poses risks to human and animal health via various exposure routes. It induces toxicity in multiple organs and disrupts reproductive health by affecting steroid hormone synthesis and fetal development. In this study, we investigated the impact of TEB on fetal testes using in vitro models, focusing on germ, Sertoli, and Leydig cells, and explored the mechanisms underlying cellular damage. The results revealed significant damage to germ cells and disruption of Leydig cell development. TEB exposure led to a decrease in germ cell numbers, as indicated by histological and immunostaining analyses. TEB induced the up- and down-regulation of the expression of fetal and adult Leydig cell markers, respectively. Additionally, TEB-treated fetal testes exhibited increased expression of oxidative-stress-related genes and proteins. However, co-treatment with the antioxidant N-acetylcysteine mitigated TEB-induced germ cell damage and prevented abnormal Leydig cell development. These findings suggest that administration of antioxidants can prevent the intratesticular damage typically caused by TEB exposure.

Atrazine disorganises laminin formation and reduces cell numbers in the tammar testis during early differentiation.

Atrazine, like oestrogen, disorganises laminin formation and reduces the number of germ cells and Sertoli cells in the developing testes of the tammar wallaby. This study suggests that interfering with the balance of androgen and oestrogen affects the integrity of laminin structure and testis differentiation. The herbicide atrazine was banned in Europe in 2003 due to its endocrine disrupting activity but remains widely used. The integrity of the laminin structure in fetal testis cords requires oestrogen signalling but overexposure to xenoestrogens in the adult can cause testicular dysgenesis. However, whether xenoestrogens affect laminin formation in developing testes has not been investigated. Here we examined the effects of atrazine in the marsupial tammar wallaby during early development and compare it with the effects of the anti-androgen flutamide, oestrogen, and the oestrogen degrader fulvestrant. The tammar, like all marsupials, gives birth to altricial young, allowing direct treatment of the developing young during the male programming window (day 20-40 post partum (pp)). Male pouch young were treated orally with atrazine (5 mg/kg), flutamide (10 mg/kg), 17β-oestradiol (2.5 mg/kg) and fulvestrant (1 mg/kg) daily from day 20 to 40 pp. Distribution of laminin, vimentin, SOX9 and DDX4, cell proliferation and mRNA expression of SRY, SOX9, AMH, and SF1 were examined in testes at day 50 post partum after the treatment. Direct exposure to atrazine, flutamide, 17β-oestradiol, and fulvestrant all disorganised laminin but had no effect on vimentin distribution in testes. Atrazine reduced the number of germ cells and Sertoli cells when examined at day 40-50 pp and day 20 to 40 pp, respectively. Both flutamide and fulvestrant reduced the number of germ cells and Sertoli cells. Atrazine also downregulated SRY expression and impaired SOX9 nuclear translocation. Our results demonstrate that atrazine can compromise normal testicular differentiation during the critical male programming window.

The ameliorative effects of curcumin nanomicelle on testicular damage in the mouse model of multiple sclerosis

BackgroundThis study investigated the effect of curcumin nanomicelle (CUR-n) on the structure of testis tissue, the process of spermatogenesis, LH, FSH, testosterone, and oxidative stress in a model of multiple sclerosis.MethodsTwenty-four male mice C57BL/6 were randomly allocated into 4 groups of 6 (1: group receiving 2% CPZ diet, 2: group receiving the diet of 2% CPZ + CUR-n with a dose of 50 mg/kg, 3: group receiving the diet of 2% CPZ + CUR-n with a dose of 100 mg/kg). The concentration of hormones (testosterone, LH and FSH), was measured by the special hormone assay ELISA kits. Measuring total antioxidant capacity (TAC) and Malondialdehyde (MDA) levels was done by spectrophotometry and calorimetric methods, respectively. Stereological analysis was done in order to explore the number of spermatogenesis cells, testis and sperm properties.ResultsThe results indicated that CUR-n (100 mg/kg) significantly enhanced the concentration of LH, FSH, testosterone, and TAC but reduced MDA levels. It also notably increased the quantity of spermatogonia, spermatocyte, round spermatids, long spermatids and LCs, augmented testis weight and volume, and germinal epithelium volume, improved sperm count, morphology, viability, and motility. In addition, a considerable decrease in the amount of wrinkling and disruption of the germinal epithelium was observed after intervention with CUR-n (100 mg/kg). Furthermore, a significant increase in the number of germ cells compared to the group receiving CPZ was detected.ConclusionThis study proposes that CUR-n could be a therapeutic agent for decreasing the adverse effects of MS on testis.

Heat stress-induced dysbiosis of the gut microbiota impairs spermatogenesis by regulating secondary bile acid metabolism in the gut

Heat stress (HS) poses a substantial challenge to livestock. Studies have demonstrated that HS reduces fertility and leads to gut microbiota dysbiosis in bulls. However, the impact of the gut microbiota on fertility in bulls during HS is still unclear. Our research revealed that HS exposure decreased semen quality in bulls, and fecal microbiota transplantation (FMT) from heat-stressed bulls to recipient mice resulted in a significant decrease in number of testicular germ cells and epididymal sperm. Untargeted metabolomics methodology and 16S rDNA sequencing conjoint analysis revealed that Akkermansia muciniphila (A. muciniphila) seemed to be a key bacterial regulator of spermatogenesis after HS exposure. Moreover, the research indicated that A. muciniphila regulated secondary bile acid metabolism by promoting the colonization of bile salt hydrolase (BSH)-metabolizing bacteria, leading to increase of retinol absorption in the host gut and subsequently elevation of testicular retinoic acid level, thereby improving spermatogenesis. This study sheds light on the relationship between HS-induced microbiota dysbiosis and spermatogenesis, offering a potential therapeutic approach for addressing bull spermatogenic dysfunction triggered by HS exposure.

Changes in characteristics of spermatogonial stem cells in response to heat stress in stallions

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions.

Prenatal nicotine exposure leads to epigenetic alterations in peripheral nervous system signaling genes in the testis of the rat

BackgroundPrenatal nicotine exposure (PNE) has been documented to cause numerous deleterious effects on fetal development. However, the epigenetic changes promoted by nicotine exposure on germ cells are still not well understood.ObjectivesIn this study, we focused on elucidating the impact of prenatal nicotine exposure on regulatory epigenetic mechanisms important for germ cell development.MethodsSprague-Dawley rats were exposed to nicotine during pregnancy and male progeny was analyzed at 11 weeks of age. Testis morphology was analyzed using frozen testis sections and expression of germ cell markers was examined by RT-qPCR; histone modifications were assessed by Western Blot (WB). DNA methylation analysis was performed by methylation-specific PCR of bisulfite converted DNA. Genome-wide DNA methylation was analyzed using Methylated DNA immunoprecipitation (MeDIP)-seq. We also carried out transcriptomics analysis of pituitary glands by RNA-seq.ResultsWe show that gestational exposure to nicotine reduces germ cell numbers, perturbs meiosis, affects the expression of germ line reprogramming responsive genes, and impacts the DNA methylation of nervous system genes in the testis. PNE also causes perturbation of gene expression in the pituitary gland of the brain.ConclusionsOur data demonstrate that PNE leads to perturbation of male spermatogenesis, and the observed effects are associated with changes of peripheral nervous system signaling pathways. Alterations in the expression of genes associated with diverse biological activities such as cell migration, cell adhesion and GABA signaling in the pituitary gland underscore the complexity of the effects of nicotine exposure during pregnancy.

Primordial germ cell DNA demethylation and development require DNA translesion synthesis

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

Histopathological study of the fertility components of undescended testes in boys of different age group

Background: Undescended testes presents the most common male developmental abnormalities, with infertility frequently observed in unilateral as well as bilateral forms. In that study, we evaluated the histological parameters of Undescended testes based on patient’s age and we recommended for the optimal timing for orchiopexy. Objective: To evaluate the histological parameters of undescended testis and determine the optimal timing for orchiopexy. Methods and Materials: This cross-sectional study included 30 patients with undescended testis(es) in the department of paediatric surgery, BSMMU. After taking relevant history, examination findings and investigation reports of all the study subjects were recorded in the pretested data collection sheet. Then biopsy specimens were collected from the selected testes during orchiopexy and analyzed the parameters including mean tubular diameter (MTD), mean tubular fertility index (MTFI) germ cell count (GCC), Sertoli cell index (SCI) and interstitial fibrosis index (IFI) Results: The MTFI and GCC in children &lt;1 years of age were significantly higher than those of other older age group. (P= 0.011 &amp; P= 0.003) The MTFI, GCC and IFI were significantly better in patients &lt;2 years of age when compared to those of &gt; 2years. IFI in the &gt; 5-years group was significantly higher than those of any of the other younger age groups (P &lt; 0.05). Other parameters such as testicular volume, MTD and SCI showed no statistically significant difference between the age groups studied. Conclusions: This study revealed that higher number of germ cells exists in the tubules between 6 months and 1 year. After first year of life, the major germ cell parameters deteriorate continuously, thus we recommended that orchipexy should be performed, between 6 months and 1 year but no later than 2 years for preserving future fertility. Journal of Paediatric Surgeons of Bangladesh (2019) Vol.10 (1 &amp; 2): 26-31

Impaired Spermatogenesis in Infertile Patients with Orchitis and Experimental Autoimmune Orchitis in Rats.

Experimental autoimmune orchitis (EAO) is a well-established rodent model of organ-specific autoimmunity associated with infertility in which the testis immunohistopathology has been extensively studied. In contrast, analysis of testis biopsies from infertile patients associated with inflammation has been more limited. In this work, testicular biopsies from patients with idiopathic non-obstructive azoospermia diagnosed with hypospermatogenesis (HypoSp) [mild: n = 9, and severe: n = 11], with obstructive azoospermia and complete Sp (spermatogenesis) (control group, C, n = 9), and from Sertoli cell-only syndrome (SCOS, n = 9) were analyzed for the presence of immune cells, spermatogonia and Sertoli cell (SCs) alterations, and reproductive hormones levels. These parameters were compared with those obtained in rats with EAO. The presence of increased CD45+ cells in the seminiferous tubules (STs) wall and lumen in severe HypoSp is associated with increased numbers of apoptotic meiotic germ cells and decreased populations of undifferentiated and differentiated spermatogonia. The SCs showed an immature profile with the highest expression of AMH in patients with SCOS and severe HypoSp. In SCOS patients, the amount of SCs/ST and Ki67+ SCs/ST increased and correlated with high serum FSH levels and CD45+ cells. In the severe phase of EAO, immune cell infiltration and apoptosis of meiotic germ cells increased and the number of undifferentiated and differentiated spermatogonia was lowest, as previously reported. Here, we found that orchitis leads to reduced sperm number, viability, and motility. SCs were mature (AMH-) but increased in number, with Ki67+ observed in severely damaged STs and associated with the highest levels of FSH and inflammatory cells. Our findings demonstrate that in a scenario where a chronic inflammatory process is underway, FSH levels, immune cell infiltration, and immature phenotypes of SCs are associated with severe changes in spermatogenesis, leading to azoospermia. Furthermore, AMH and Ki67 expression in SCs is a distinctive marker of severe alterations of STs in human orchitis.

Photoperiod-regulated mitophagy in the germ cells of Brandt's voles (Lasiopodomys brandtii).

Photoperiod is a pivotal factor in affecting testicular function and spermatogenesis in seasonal-breeding animals. Mitophagy is essential for spermatogenesis, but its association with seasonal photoperiods has not been studied extensively. To explore this, we exposed male Brandt's voles (Lasiopodomys brandtii) to long-photoperiod (LP, 16 h/day) and short-photoperiod (SP, 8 h/day) conditions from their embryonic stages. Our results indicated that testis weight, volume, and relative testes weight were all significantly increased in LP compared to SP. Additionally, blood testosterone levels were markedly higher in LP than SP. Histological examination revealed that seminiferous diameter and epithelium thickness were greater in LP, with an increased abundance of germ cell types and cell numbers compared to SP. RT-qPCR analysis showed that mitophagy-promoting genes, such as Pink1, Prkn, Tomm7, Mnf2, Lc3, Optn, Gabarap, and Nbr1 were all upregulated in LP. Fluorescence in situ hybridization indicated that Pink1 expression was present in spermatogonia in SP, while in LP, Pink1 expression extended to almost all germ cell types with significantly higher mean optical density. Prkn expression was found in all germ cell types in both LP and SP, with a significantly higher mean optical density of 10-week-old LP males. Transmission electron microscopy showed normal mitochondrial morphology with clear membranes in SP, while the LP group had reduced cristae in mitochondria and damaged mitochondria undergoing autophagy. This study suggests that mitophagy may be involved in the photoperiodic spermatogenesis in Brandt's voles, providing insights into the role of photoperiod in seasonal reproduction in wild animals.

JAK-STAT-dependent contact between follicle cells and the oocyte controls Drosophila anterior-posterior polarity and germline development

The number of embryonic primordial germ cells in Drosophila is determined by the quantity of germ plasm, whose assembly starts in the posterior region of the oocyte during oogenesis. Here, we report that extending JAK-STAT activity in the posterior somatic follicular epithelium leads to an excess of primordial germ cells in the future embryo. We show that JAK-STAT signaling is necessary for the differentiation of approximately 20 specialized follicle cells maintaining tight contact with the oocyte. These cells define, in the underlying posterior oocyte cortex, the anchoring of the germ cell determinant oskar mRNA. We reveal that the apical surface of these posterior anchoring cells extends long filopodia penetrating the oocyte. We identify two JAK-STAT targets in these cells that are each sufficient to extend the zone of contact with the oocyte, thereby leading to production of extra primordial germ cells. JAK-STAT signaling thus determines a fixed number of posterior anchoring cells required for anterior-posterior oocyte polarity and for the development of the future germline.

An in vitro three-dimensional (3D) testicular organoid culture system for efficient gonocyte maintenance and propagation using frozen/thawed neonatal bovine testicular tissues

Fertility preservation in prepubertal boys with cancer requires the cryopreservation of immature testicular tissues (ITTs) prior to gonadotoxic treatment. However, the limited number of germ cells in small human ITT biopsies necessitates the development of an in vitro culture system for germ cell expansion using frozen-thawed ITTs. Here, we generated testicular organoids for the in vitro maintenance and expansion of gonocytes from frozen-thawed two-week-old neonatal bovine ITTs. We investigated the effects of different cell-seeding densities, culture serums, seeding methods, and gonadotropin supplementations, on the maintenance and proliferation of enriched gonocytes. Our results demonstrated that enriched gonocytes and testicular cells from frozen-thawed neonatal ITTs could self-assemble into spheroid organoids in three days in an appropriate Matrigel-based culture environment. For the optimal formation of prepubertal testicular organoids, a seeding density of 1 × 106 cells/well is recommended over other densities. This strategy results in organoids with a mean diameter of 60.53 ± 12.12 μm; the mean number of organoids was 5.57 ± 1.60/105 μm2 on day 11. The viability of organoids was maintained at 79.75 ± 2.99% after being frozen and thawed. Supplementing the culture medium with glial cell-derived neurotrophic factor, fibroblast growth factor 2, and leukemia inhibitory factor, increased the proportion of KI67-positive proliferating cells in organoids, elevated the expression of C-KIT but reduced the expression of GFRα1 at day 28 when compared to those without hormone supplements (p< 0.05). In addition, supplementing the culture medium with follicle-stimulating hormone and testosterone helped to maintain a significantly higher viability (p< 0.05) in ITT organoids at day 28. These organoids could be cryopreserved for storage and thawed as needed. The successful generation of ITT organoids provides a valuable tool for establishing in vitro spermatogenesis, propagating human germ cells, investigating testicular physiology and the origin of germ cell tumors, and testing the toxicity of new drugs in future clinical applications.