Exosomes released from Sertoli cells contribute to the survival of Leydig cells through CCL20 in rats.

Reciprocal communication between Sertoli and Leydig cells occurs in the testes; however, the detailed mechanisms involved are not completely understood. Exosomes can communicate within neighboring or distant cells to regulate cell function. Our aim was to determine whether exosomes released from Sertoli cells can regulate the survival of Leydig cells. We found that exosomes released from rat primary Sertoli cells could be internalized by Leydig cells in vitro, and promote the survival of Leydig cells, as assessed by optical density at 450 nm, compared to untreated control (mean ± SD: 0.95 ± 0.04 vs 0.79 ± 0.03, P < 0.05). When the exosomes were injected into the interstitial area of rat testis, they could also be internalized by Leydig cells in vivo. To investigate if exosomes released from Sertoli cells can reach Leydig cells in vivo, exosomes were injected into the efferent duct, from where they entered the interstitial space from seminiferous tubules, which indicated that they may cross the blood-testis barrier (BTB). Further in vitro studies found that exosomes released from Sertoli cells significantly increased CC-chemokine ligand 20 (Ccl20) mRNA (mean ± SD: 2.79 ± 0.08 vs 0.98 ± 0.04, P < 0.01) and protein (mean ± SD: 1.08 ± 0.06 vs 0.53 ± 0.05 ng/ml, P < 0.01) levels in Leydig cells, compared to the untreated Leydig cells. CCL20 promoted the phosphorylation of AKT (protein kinase B) in Leydig cells, compared to untreated control (mean ± SD: 0.074 ± 0.002 vs 0.051 ± 0.002, P < 0.01). In conclusion, our results demonstrated that exosomes released by Sertoli cells may cross the BTB and promote the survival of Leydig cells. The findings may add new evidence for Sertoli-Leydig cell communication.

Comparison of intracytoplasmic sperm injection (ICSI) outcomes in infertile men with spermatogenic impairment of differing severity.

The extent of spermatogenic impairment on intracytoplasmic sperm injection (ICSI) outcomes and the risk of major birth defects have been little assessed. In this study, we evaluated the relationship between various spermatogenic conditions, sperm origin on ICSI outcomes, and major birth defects. A total of 934 infertile men attending the Center for Reproductive Medicine of Ren Ji Hospital (Shanghai, China) were classified into six groups: nonobstructive azoospermia (NOA; n = 84), extremely severe oligozoospermia (esOZ; n = 163), severe oligozoospermia (sOZ, n = 174), mild oligozoospermia (mOZ; n = 148), obstructive azoospermia (OAZ; n = 155), and normozoospermia (NZ; n = 210). Rates of fertilization, embryo cleavage, high-quality embryos, implantation, biochemical and clinical pregnancies, abortion, delivery, newborns, as well as major birth malformations, and other newborn outcomes were analyzed and compared among groups. The NOA group showed a statistically lower fertilization rate (68.2% vs esOZ 77.3%, sOZ 78.0%, mOZ 73.8%, OAZ 76.6%, and NZ 79.3%, all P < 0.05), but a significantly higher implantation rate (37.8%) than the groups esOZ (30.1%), sOZ (30.4%), mOZ (32.6%), and OAZ (31.0%) (all P < 0.05), which was similar to that of Group NZ (38.4%). However, there were no statistically significant differences in rates of embryo cleavage, high-quality embryos, biochemical and clinical pregnancies, abortions, deliveries, major birth malformations, and other newborn outcomes in the six groups. The results showed that NOA only negatively affects some embryological outcomes such as fertilization rate. There was no evidence of differences in other embryological and clinical outcomes with respect to sperm source or spermatogenic status. Spermatogenic failure and sperm origins do not impinge on the clinical outcomes in ICSI treatment.

Seminal exosomal miR-210-3p as a potential marker of Sertoli cell damage in Varicocele.

BACKGROUND: Varicocoele-associated stressors, such as hypoxia and heat, can damage cell function and viability, and some exosomal biomarkers released from impaired cells may reflect the cell status in testis. OBJECTIVES: To find if seminal exosomal microRNAs can reflect the Sertoli cell function in varicocoele. MATERIALS AND METHODS: Experimental left varicocoele rat model was established (n = 24), and patients with different grades of varicocoele (n = 104) were enrolled. Primary rat Sertoli cells were isolated with enzymatic hydrolysis. Exosomes were isolated from primary rat Sertoli cells, rat epididymis tissue, and human seminal plasma with polymer-based precipitation method. Exosomal microRNAs were quantified with qPCR. Inhibin-B was detected with enzyme immunoassay. The correlation analysis between microRNA and inhibin-B was evaluated with Spearman's correlation. RESULTS: We screened 12 previously reported hypoxia-responsive microRNAs in the primary rat Sertoli cells and found that 4 exosomal microRNAs increased significantly in response to in vitro hypoxia treatment (P < .05). Of the 4 microRNAs, only miR-210-3p was upregulated in the rats with experimental varicocoele (P < .01). In the patients with varicocoele, we found that seminal exosomal miR-210-3p significantly increased in patients with grade II and III varicocoele (P < .01), and miR-210-3p negatively correlated with sperm count (P < .01) and seminal inhibin-B expression (r = -0.39, P < .01). For the 30 patients with microsurgical varicocelectomy, the operation notably decreased miR-210-3p (P < .01). DISCUSSION AND CONCLUSION: Seminal exosomal miR-210-3p may be a novel, sensitive, and non-invasive biomarker of Sertoli cell damage in varicocoele.

A novel micro-straw for cryopreservation of small number of human spermatozoon.

Cryopreservation of few spermatozoa is still a major challenge for male fertility preservation. This study reports use a new micro-straw (LSL straw) for freezing few spermatozoa for intracytoplasmic sperm injection (ICSI). Semen samples from 22 fertile donors were collected, and each semen sample was diluted and mixed with cryoprotectant in a ratio of 1:1, and then frozen using three different straws such as LSL straw (50-100 µl), traditional 0.25 ml and 0.5 ml straws. For freezing, all straws were fumigated with liquid nitrogen, with temperature directly reducing to -130--140°C. Sperm concentration, progressive motility, morphology, acrosome integrity, and DNA fragmentation index were evaluated before and after freezing. After freezing-thawing, LSL straw group had significantly higher percentage of sperm motility than traditional 0.25 ml and 0.5 ml straw groups (38.5% vs 27.4% and 25.6%, P < 0.003). Sperm motility and acrosomal integrity after freezing-thawing were significantly lower than that of before freezing. However, there was no significant difference in morphology, acrosome, and DNA integrity between the three types of straws (P > 0.05). As LSL straws were thinner and hold very small volume, the freezing rate of LSL straw was obviously faster than 0.25 ml straw and 0.5 ml straws. In conclusion, LSL micro-straws may be useful to store few motile spermatozoa with good recovery of motility for patients undergoing ICSI treatment.

Association of a TDRD1 variant with spermatogenic failure susceptibility in the Han Chinese.

PURPOSE: Piwi-interacting RNAs (piRNAs) are a broad group of noncoding small RNAs that have important biological functions in germline cells and can maintain genome integrity via silencing of retrotransposons. In this study, we aimed to explore the associations between genetic variants of important genes involved in piRNA biogenesis and male infertility with spermatogenic impairment. METHODS: To this end, five single-nucleotide polymorphisms (SNPs) in the ASZ1, PIWIL1, TDRD1, and TDRD9 genes were genotyped by TaqMan allelic discrimination assays in 342 cases of nonobstructive azoospermia (NOA) and 493 controls. RESULTS: The SNP rs77559927 in TDRD1 was associated with a reduced risk of spermatogenic impairment. The genotypes TC and TC + CC showed odds ratios and 95 % confidence intervals of 0.73 (0.55-0.98, P = 0.034) and 0.73 (0.56-0.97, P = 0.030), respectively, in patients with NOA compared with those in the controls. CONCLUSION: Thus, our results provided the first epidemiological evidence supporting the involvement of TDRD1 genetic polymorphisms in piRNA processing genes in determining the risk of spermatogenic impairment in a Han Chinese population.

[Pre-freezing equilibration improves post-thaw human semen parameters].

OBJECTIVE: To investigate the effect of pre-freezing equilibration on the cryo-survival of human sperm and to optimize the protocol of direct fumigation for the freeze-thawing of human sperm. METHODS: We collected 50 semen samples from healthy donors, each subjected to cryopreservation with 3 different methods: non-equilibration freezing (Group A), 10-min equilibration at room temperature before freezing (Group B), and 10-min equilibration at 4 degrees C before freezing (Group C). We examined all the post-thaw semen samples by computer-assisted semen analysis for the sperm motility parameters, and detected the sperm vitality and deformity index (SDI). RESULTS: The recovery rate of progressive sperm motility was (61.88 +/- 16.94)% in Group C, remarkably higher than in A ([48.61 +/- 16.44]%) and B ([49.41 +/- 13.77]%) (P < 0.05), but with no significant difference between the latter two. And there were no significant differences in sperm vitality and SDI among the three groups. CONCLUSION: Ten-minute equilibration at 4 degrees C before freezing can evidently improve the progressive motility of sperm in addition to its advantages of easy operation and controllable experimental condition.

[Desert hedgehog regulates the proliferation and differentiation of Leydig cells: an update].

Hedgehog signaling pathway plays an important role in regulating the development of endocrine glands. Desert hedgehog (Dhh) has become a recent focus for its regulation of testis development, especially of Leydig cells. Dhh, as a Sertoli cell product, regulates the proliferation and differentiation of Leydig cell lineage and functions to secrete testosterone through a paracrine signaling mechanism. Testosterone, as the most important sex hormone of the male, plays a critical role in testis development, spermatogenesis and maintenance of normal masculinization. Therefore, normal Dhh signaling pathway ensures normal spermatogenic function. Researches on the Hedgehog signaling pathway in the testis have a potential significance for studying the pathophysiological mechanisms of androgen deficiency and dyszoospermia, as well as for the clinical treatment of these diseases.

[Reconstructed seminiferous tubules from ectopic grafting of testicular cells].

OBJECTIVE: To investigate the ectopic grafts of mouse testicular cells by observing the reconstruction of seminiferous tubules, colonization of spermatogenic cells and spermatogenesis using immunodeficient mice as recipients. METHODS: The testes of newborn male ICR mice were digested to obtain single cell suspension. The cells were then mixed with matrigel and subcutaneously grafted into the dorsal region of the male nude mice. The mice were castrated after the operation and the grafts were dissected from 5 of the nude mice at 4, 6, 8 and 10 weeks, respectively. The success rates of transplantation and the graft diameters were calculated, and the structure of the reconstituted seminiferous tubules, colonization of the germ cells and spermatogenesis were observed by HE staining and immunohistochemistry. RESULTS: All the mice recipients survived after the testicular cell transplantation. Within 10 weeks after the operation, tissue masses could be observed, with the diameter increased from (3.91 +/- 0.71) mm at 4 weeks to (6.69 +/- 0.50) mm. Neovascularization was detected at the surface of the masses and seminiferous tubule structures found in the grafts. The germ cells that developed from spermatogonia to round spermatids were observed, but with no sperm in the tubules. Germ cells, Sertoli cells and Leydig cells were identified by immunochemical detection of Mvh, Gata4 and P450Scc in the grafts at 8 weeks. CONCLUSION: Seminiferous tubules could be ectopically reconstructed from suspension of neonatal mouse testicular cells. Ectopic grafting provided a preferable model for the studies on testis tissue engineering and interactions between testicular cells during testicular development and spermatogenesis.