Complete Spermatogenesis Research Articles

Genetic expansion of chaperonin-containing TCP-1 (CCT/TRiC) complex subunits yields testis-specific isoforms required for spermatogenesis in planarian flatworms.

Chaperonin-containing Tail-less complex polypeptide 1 (CCT) is a highly conserved, hetero-oligomeric complex that ensures proper folding of actin, tubulin, and regulators of mitosis. Eight subunits (CCT1-8) make up this complex, and every subunit has a homolog expressed in the testes and somatic tissue of the planarian flatworm Schmidtea mediterranea. Gene duplications of four subunits in the genomes of S. mediterranea and other planarian flatworms created paralogs to CCT1, CCT3, CCT4, and CCT8 that are expressed exclusively in the testes. Functional analyses revealed that each CCT subunit expressed in the S. mediterranea soma is essential for homeostatic integrity and survival, whereas sperm elongation defects were observed upon knockdown of each individual testis-specific paralog (Smed-cct1B; Smed-cct3B; Smed-cct4A; and Smed-cct8B), regardless of potential redundancy with paralogs expressed in both testes and soma (Smed-cct1A; Smed-cct3A; Smed-cct4B; and Smed-cct8A). Yet, no detriment was observed in the number of adult somatic stem cells (neoblasts) that maintain differentiated tissue in planarians. Thus, expression of all eight CCT subunits is required to execute the essential functions of the CCT complex. Furthermore, expression of the somatic paralogs in planarian testes is not sufficient to complete spermatogenesis when testis-specific paralogs are knocked down, suggesting that the evolution of chaperonin subunits may drive changes in the development of sperm structure and that correct CCT subunit stoichiometry is crucial for spermiogenesis.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue.

Testicular cytology by fine needle aspiration in domestic cats

In cases where semen collection in tom-cats is not possible, FNA of testes is the alternative to evaluate sperm production. Although this technique for the diagnosis of fertility problems has been well discussed in other mammals (men, dogs, stallions), data for domestic cats are limited. Therefore, the aim of this study was to verify the reliability of FNA using needles of small diameters (22G and 29G) in testes of domestic cats of different ages to assess the spermatogenesis status and to present description of germ cells and Sertoli cells for cytological examinations. Thirty-four mixed breed cats aged between four months and two years presented for neutering to a Veterinary Hospital were used in this study. Under general anesthesia, testicular measures and FNA were followed by orchiectomy and imprints of the parenchyma of testes and epididymides. Cats were assigned into 3 groups: (G1) 10 cats aged less than 6 months, (G2) 14 cats aged between 6 months and one year and (G3) 10 cats aged more than one year. Cats weighted between 1.5 and 6.0 kg. The mean testicular volume (TV cm3) was 0.55 (G1), 1.18 (G2) and 2.66 (G3). Hemorrhages in the needle path were observed in more than 70% of testes. Few samples (4/68) were excluded due to blood contamination. All germ cells and Sertoli cells were identified and quantified in imprint and FNA smears. Incomplete spermatogenesis was observed in cats aged less than 6 months using both techniques (FNA and imprint); therefore, testicular FNA should not be recommended for cats at this age. Complete spermatogenesis was found in 64% of cats aged from 6 months up to one year and in all cats aged more than one year. There were no differences of Sertoli cell Index (SEI) and Spermatic Index (SI) between FNA and imprints of cats older than 6 months. In conclusion, FNA using needles of small diameter in the testes of domestic cats is viable, reliable and can be used as a tool for the analysis of the spermatogenesis status of cats older than 6 months, mainly in cases in which semen collection is not possible.

Andrology of male-to-female transsexuals: influence of cross-sex hormone therapy on testicular function.

Patients with gender dysphoria are offered cross-sex hormone therapy and sex reassignment surgery to achieve the transition between the sex assigned at birth and gender identity. According to international guidelines, cross-sex hormone therapy in trans-women should lead to a psychologically and physiologically healthy body with feminized serum hormone levels, resulting in suppression of spermatogenesis. However, in a recently published multi-center study, we discovered a high proportion of patients with male serum hormone levels and qualitatively intact spermatogenesis on the day of sex reassignment surgery. The objective of this study was to review the content of 11 publications that focus on the influence of cross-sex hormone therapy on testicular morphology. These publications were identified based on a PubMed search for the key words transgender/transsexual/gender dysphoria in male-to-female persons, cross-sex hormone therapy, and testicular tissues. Whereas three publications described a marked reduction of the spermatogenic level in all patients examined, eight publications reported inconsistent results. Histological analyses showed highly variable outcomes from qualitatively normal spermatogenesis and undisturbed Leydig/Sertoli cell morphology to full testicular regression with severe cellular damage and hyalinization. Explanations for these heterogeneous findings include insufficient cross-sex hormone therapy regarding dosage or duration. As complete spermatogenesis is associated with virilized serum hormone levels, these patients may face challenges especially after sex reassignment surgery in adjusting to the abruptly established hypogonadal state following removal of the testes. These findings also suggest that contraception should be discussed, and fertility preservation should be offered during/prior to cross-sex hormone therapy. There is a need for more individualized and better-controlled cross-sex hormone therapy and post-treatment regimens. Evidence-based guidelines for attending clinicians need to be established in order to deliver the most appropriate care.

Histological changes in pituitary-testes axis in rats exposed to cadmium chloride: Protective role of Eruca sativa seeds

The present study was carried out to investigate the protective effect of Eruca sativa seeds against changes of pituitary and testes in cadmium treated rats. Forty adult male albino rats were used and divided into 4 equal groups and treated for 8 weeks as follow: The first group was given distilled water and used as a control group. The second group (G1) was administered tap water containing 30 ppm/ L of cadmium chloride. Animals in third group (G2) received tap water containing Cadmium chloride as in G1 plus 250 mg/Kg B.W ethanolic extract of Eruca sativa seeds, while rats in fourth group (G3) administered 250 mg/Kg B.W of ethanolic extract of Eruca sativa seeds only. After 8 weeks of experiment the animals were anesthetized, the pituitary and testes were excised for histological studies. Results showed that cadmium chloride intake caused severe alterations in the pituitary gland manifested by a marked apoptotic cells of pars distalis, accompanied with marked necrosis leading to left large multiple spaces in their parenchyma. Whereas testes revealed shrinkage, irregular arrangement of seminiferous tubules and increase intertubular spaces. A considerable changes was also found in the seminiferous tubules with loss most of their epithelial layer and filled with cellular debris, incomplete spermatogenesis, congested blood vessels and few Leydig's cells. Administration of Eruca sativa extract reduce the deleterious effects of cadmium chloride on pituitary gland as well as more or less complete spermatogenesis process with thickness of interstitial tissue in most of seminiferous tubules. It could be suggested that the antioxidant properties of Eruca sativa extract protects the deleterious histological damage induced by cadmium chloride in adult rats.

Testicular growth and development in puberty.

To describe pubertal testicular growth in humans, changes in testicular cell populations that result in testicular growth, and the role of testosterone and gonadotrophins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in testicular growth. When human data were not available, studies in nonhuman primates and/or rodents were used as surrogates. Testicular growth in puberty follows a sigmoidal growth curve, with a large variation in timing of testicular growth and adult testicular volume. Testicular growth early in puberty is due to increase in Sertoli cell number and length of seminiferous tubules, whereas the largest and fastest growth results from the increase in the diameter of the seminiferous tubules first due to spermatogonial proliferation and then due to the expansion of meiotic and haploid germ cells. FSH stimulates Sertoli cell and spermatogonial proliferation, whereas LH/testosterone is mandatory to complete spermatogenesis. However, FSH and LH/testosterone work in synergy and are both needed for normal spermatogenesis. Testicular growth during puberty is rapid, and mostly due to germ cell expansion and growth in seminiferous tubule diameter triggered by androgens. Pre-treatment with FSH before the induction of puberty may improve the treatment of hypogonadotropic hypogonadism, but remains to be proven.

Comparison of global gene expression profiles of microdissected human foetal Leydig cells with their normal and hyperplastic adult equivalents

Do human adult Leydig cells (ALCs) within hyperplastic micronodules display characteristics of foetal LCs (FLCs)? The gene expression profiles of FLCs and all ALC subgroups were clearly different, but there were no significant differences in expressed genes between the normally clustered and hyperplastic ALCs. LCs are the primary androgen producing cells in males throughout development and appear in chronologically distinct populations; FLCs, neonatal LCs and ALCs. ALCs are responsible for progression through puberty and for maintenance of reproductive functions in adulthood. In patients with reproductive problems, such as infertility or testicular cancer, and especially in men with high gonadotrophin levels, LC function is often impaired, and LCs may cluster abnormally into hyperplastic micronodules (defined as clusters of >15 LCs in a cross-section). A genome-wide microarray study of LCs microdissected from human foetal and adult tissue samples (n=12). Additional tissue specimens (n=15) were used for validation of the mRNA expression data at the protein level. Frozen human tissue samples were used for the microarray study, including morphologically normal foetal (gestational week 10-11) testis samples, and adult testis specimens with normal LC distribution, LC micronodules or LC micronodules adjacent to hCG-producing testicular germ cell tumours. Transcriptome profiling was performed on Agilent whole human genome microarray 4×44 K chips. Microarray data pre-processing and statistical analysis were performed using the limma R/Bioconductor package in the R software, and differentially expressed genes were further analysed for gene set enrichment using the DAVID Bioinformatics software. Selected genes were studied at the protein level by immunohistochemistry. The transcriptomes of FLCs and ALCs differed significantly from each other, whereas the profiles of the normally clustered and hyperplastic ALCs were similar despite morphological heterogeneity. The study revealed several genes not known previously to be expressed in LCs during early development, including sulfotransferase family 2A member 1 (SULT2A1), WNT1-inducible signalling pathway protein 2 (WISP2), hydroxyprostaglandin dehydrogenase (HPGD) and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1), whose expression changes were validated at the protein level. The transcriptomic data are deposited in ArrayExpress (accession code E-MTAB-5453). The small number of biological replicates and the necessity of RNA amplification due to the scarcity of human tissues, especially foetal specimens, are the main limitations of the study. Heterogeneous subpopulations of LCs within micronodules were not discriminated during microdissection and might have affected the expression profiling. The study was constrained by the lack of availability of truly normal controls. Testis samples used as 'controls' displayed complete spermatogenesis and were from patients with germ cell neoplasia but with undetectable hCG and normal hormone levels. The changes in LC morphology and function observed in patients with reproductive disorders possibly reflect subtle changes in the expression of many genes rather than regulatory changes of single genes or pathways. The study provides new insights into the development and maturation of human LCs by the identification of a number of potential functional markers for FLC and ALC. The study was supported by research grants from the Danish Cancer Society, the Capital Region's Research Fund for Health Research, Rigshospitalet's research funds, the Villum Kann Rasmussen Foundation, the Danish Innovation Fund, ReproUnion, Kirsten and Freddy Johansen's foundation and the Novo Nordisk Foundation. None of the funding agencies had any influence on the study. The authors declare no conflicts of interest.

DNA methylation imprinting errors in spermatogenic cells from maturation arrest azoospermic patients.

Imprinting errors have been described in spermatozoa from infertile patients with oligozoospermia and azoospermia. However, little is known about methylation of imprinted genes in other spermatogenic cells from azoospermic patients. Therefore, we aimed to evaluate the methylation status of single CpGs located in the differentially methylated regions (DMRs) of two imprinted genes, one paternally (H19) and one maternally (MEST) methylated, in primary spermatocytes of azoospermic patients presenting complete (MAc, n=7) and incomplete (MAi, n=8) maturation arrest, as well as in other spermatogenic cells from MAi patients that presented focus of complete spermatogenesis in some seminiferous tubules. We observed H19 imprinting errors in primary spermatocytes from one MAi patient and MEST imprinting errors in one MAi and two MAc patients. Additionally, H19 imprinting errors were observed in elongated spermatids/spermatozoa from one MAi patient. Nevertheless, no statistical differences were found for H19 and MEST global methylation levels (percentage of methylated and unmethylated CpGs, respectively) between patients with complete and incomplete MA and also between MA groups and a control group. These results provide further evidence that imprinting errors occur in spermatogenic cells from patients presenting impaired spermatogenesis, as we and others have previously described in ejaculated and testicular spermatozoa. As paternal imprinting errors can be transmitted to the embryo by the sperm cell, they can provide a possible explanation for poor embryo development and/or low pregnancy rates as correct expression of imprinted genes is crucial for embryo and placental development and function. Therefore, in cases with male factor infertility where unsuccessful invitro fertilization (IVF) treatments are recurrent, analysis of imprinting marks in spermatozoa might be a useful diagnostic tool.

Update on fertility restoration from prepubertal spermatogonial stem cells: How far are we from clinical practice?

Fertility preservation in prepubertal boys facing gonadotoxic treatment is still at the experimental stage. Nevertheless cryopreservation of immature testicular tissue (ITT) obtained by small testicular biopsy is being increasingly proposed in reproductive care clinics for this purpose. Different approaches to in vivo or in vitro mature spermatogonial stem cells (SSCs) contained in ITT have been studied: autografting of testicular tissue pieces, transplantation of one's own purified germ cell suspensions, and in vitro maturation (IVM) for subsequent use of sperm for intra cytoplasmic sperm injection (ICSI). While complete spermatogenesis yielding fertile offspring has been achieved in a number of animal species after cell and tissue transplantation and IVM, no mature sperm has yet been obtained from human prepubertal SSCs. This review describes research conducted by our team and a number of others working on fertility restoration from SSCs, with special emphasis on debated concerns and progress made towards clinical application of different strategies.

Primary Human Testicular Cells Self-Organize into Organoids with Testicular Properties.

SummarySo far, successful de novo formation of testicular tissue followed by complete spermatogenesis in vitro has been achieved only in rodents. Our findings reveal that primary human testicular cells are able to self-organize into human testicular organoids (TOs), i.e., multi-cellular tissue surrogates, either with or without support of a biological scaffold. Despite lacking testis-specific topography, these mini-tissues harbored spermatogonia and their important niche cells, which retained specific functionalities during long-term culture. These observations indicate the posibility of in vitro re-engineering of a human testicular microenvironment from primary cells. Human TOs might help in the development of a biomimetic testicular model that would exert a tremendous impact on research and development, clinical treatment of infertility, and screening in connection with drug discovery and toxicology.

Spermatogenesis in Explanted Fetal Mouse Testis Tissues.

We previously reported the successful induction of complete spermatogenesis of mice in neonatal testis tissues cultured on agarose gel, with the culture medium supplemented with a bovine serum albumin product, AlbuMAX. This method, however, has not been examined for fetal testis tissues. In this report, we tested the culture method for fetal testes of the Acrosin (Acr)-Gfp transgenic mouse, whose testicular germ cells express GFP from the midmeiotic phase onward, using AlbuMAX-containing medium. The fetal testis, from 19.5 days postcoitum (dpc) back to 14.5 dpc, showed spermatogenic progression and produced haploid cells in culture. On the other hand, testes of 13.5 dpc or earlier did not show the meiotic sign of Acr-Gfp expression. Regardless of the fetal age, tissue masses enlarged during the culture period because of the elongation and thickening of the seminiferous tubules. This simple culture method could be a useful experimental system to investigate fetal testicular development and germ cell biology.

Predictors of spermatogenesis in radical orchiectomy specimen and potential implications for patients with testicular cancer

To assess the ability of semen analysis and other patients' characteristics to predict the presence of spermatozoa in radical orchiectomy pathological specimen, and describe potential implications for patients with azoospermia and testis cancer. Retrospective cohort study. Tertiary hospital. A total of 214 consecutive patients with testicular cancer who underwent radical orchiectomy between 1997 and2015. None. Histologic slides were reviewed and the presence of mature spermatozoa was documented. Clinical, laboratory, and radiographic characteristics were recorded. Logistic regression analyses were used to identify factors associated with the presence of spermatozoa in the noninvolved ipsilateral testicular parenchyma. Spermatozoa were found in the pathological specimen of 145 patients (67.8%). At multivariate analysis, increased tumor size was the only factor associated with lower rates of spermatozoa in the specimen. Mean tumor diameter was 4.06cm, and spermatozoa were found in 83% and 49% of testes with tumor diameters <4 and ≥4cm, respectively. Preoperative semen analysis records were available for 107 patients. Oligozoospermia, severe oligozoospermia, azoospermia, and cryptozoospermia were observed in 17 (16%), 18 (17%), 9 (8%) and 3 (3%) patients, respectively. Sperm concentration and motility were not associated with complete spermatogenesis. Seven of 12 patients (58%) with either azoospermia or cryptozoospermia had mature sperm in their pathological sections. Larger testicular cancers are associated with lower rates of spermatozoa in the ipsilateral testis. Given the substantial likelihood (∼60%) of spermatozoa to be present in the cancerous testis of patients with azoospermia and cryptozoospermia, concomitant oncologic testicular sperm extraction (TESE) can be considered in these selected patients.

Knockout of BRD7 results in impaired spermatogenesis and male infertility.

BRD7 was originally identified as a novel bromodomain gene and a potential transcriptional factor. BRD7 was found to be extensively expressed in multiple mouse tissues but was highly expressed in the testis. Furthermore, BRD7 was located in germ cells during multiple stages of spermatogenesis, ranging from the pachytene to the round spermatid stage. Homozygous knockout of BRD7 (BRD7−/−) resulted in complete male infertility and spermatogenesis defects, including deformed acrosomal formation, degenerative elongating spermatids and irregular head morphology in postmeiotic germ cells in the seminiferous epithelium, which led to the complete arrest of spermatogenesis at step 13. Moreover, a high ratio of apoptosis was determined by TUNEL analysis, which was supported by high levels of the apoptosis markers annexin V and p53 in knockout testes. Increased expression of the DNA damage maker λH2AX was also found in BRD7−/− mice, whereas DNA damage repair genes were down−regulated. Furthermore, no or lower expression of BRD7 was detected in the testes of azoospermia patients exhibiting spermatogenesis arrest than that in control group. These data demonstrate that BRD7 is involved in male infertility and spermatogenesis in mice, and BRD7 defect might be associated with the occurrence and development of human azoospermia.

A case of intersex occurrence in Steindachneridion parahybae (Steindachner, 1877) (Siluriformes: Pimelodidae) under captivity condition: a cytogenetic and morphological study

ABSTRACT Little is known about reproductive biology of endangered Steindachneridion parahybae , a gonochoristic teleost species inhabiting the Paraíba do Sul River Basin, and herein is the first description of intersex in S. parahybae juvenile. The normal appearance of ovaries and testes in juvenile from the same lot of breeding were also described for comparison, even as cytogenetic analysis was performed in these juveniles. One specimen was a priori classified as female due to the macroscopic characteristic of ovaries, with small yellow oocytes, without fringes (a main characteristic of catfish male), and larger than testes; however the microscopic analysis revealed the presence of ovotestes, including the complete spermatogenesis. S. parahybae had diploid number, 2n = 56 chromosomes with no evidence of differentiated sex chromosomes or supernumerary chromosomes among them. These findings may be due to the result of exposure to endocrine disrupting compounds or may also be influenced by environmental conditions. The possibility of intersexes might also happen spontaneously and it cannot be ruled out. Therefore, the functional significance and reproductive consequences of this anomaly remain to be determined, suggesting that this species may be susceptible to endocrine disruption. These results contribute to gain expertise about reproductive biology of an endangered species in captivity.

Role of alcoholic extract of Roket (Eruca sativa) leaves on male reproduction of experimentally induced-oxidative stressed rats

This study conducted to find out the protective role of ethanolic extract of Eruca sativa leaves against the deleterious effect of hydrogen peroxide on some aspect of male reproduction in adult rats. Forty adult male rats were randomly assigned into four equal groups as follows: control group received tap water (untreated); group T1 were received tap water containing 0.5% H2O2; group T2 were received tap water containing 0.5% H2O2 plus administration of 300 mg/kg. B.W. ethanolic extract of Eruca sativa leaves and group T3 administration ethanolic extract of Eruca sativa leaves only at the same dose of group T2. All treatments continued for 60 days. At the end of the experiment, samples of tests and epididymis tissues were taken to prepare histological sections for measurement the diameter of seminiferous tubules, thickness of epithelial cells of seminiferous tubules and histological examination of testes and epididymis. The results in Group T1 showed a significant decrease in the diameter and thickness of epithelial cells of seminiferous tubules, but these parameters clarified a significant increase in T2 and T3 groups as compared with T1 group. Histological sections of testis and epididymis in group T1 revealed incomplete spermatogenesis, cell debris, vacculation of Sertoli cells and view sperms in the lumen of seminiferous tubules and epididymis. Besides, normal obvious histological architecture of seminiferous tubules and epididymis with complete spermatogenesis were shown in sections of testis and epididymis of T2 and T3 groups' as compared to T1. In conclusion, hydrogen peroxide may impair spermatogenesis, furthermore, the results confirm the protective role of E. sativa leaves extract against oxidative stress induced by H2O2 in rats.

Causes and Factors of Male Infertility

The preservation of reproductive health of the population is an important factor of demographic policy of the state. According to some authors from 14 to 30% of couples of reproductive age suffer from infertility, malefactor in such marriages is detected in more than half of the cases. As you know, in recent years there has been a significant deterioration in the main indicators of reproductive function of men. Increased the number of andrological diseases, morphological disorders of the male reproductive system, almost halved the production of sperm in men of reproductive age. The reason probably lies behind a whole range ofstressfactors, such as medical ignorance, uncontrolled and inappropriate use of medication, metabolic disturbances, lack of vitamins and minerals, the impact of industrial pollutants, as well as the growth of addictive disorders (alcoholism, smoking and drug addiction). The forms of infertility differ according to its etiology and severity from minor changes to complete spermatogenesis dysfunction of the gonads, and can also occur due to genetic disorders. The lack of analysis of the relationship between clinical and genetic-biochemical components in men with infertility makes it impossible to understand the pathogenesis of infertility and to assess the risks of male infertility. High level of current medicine does not always guarantee an identification of the cause of male infertility. The article analyzes data from the review of specialized literature on the diagnosis and etiopathogenesis of male infertility. Frequency and clinical signs of pathology of the male reproductive system depend on the combinatorial effects of environmental influences, manifested most often in mutually reinforcing effect. A combination of several, seemed to be imperceptible factors makes the risk of development of male reproductive pathology very high. This situation compels specialists to conduct comprehensive studies on the men reproductive potential.

Putting the brakes on reproduction: Implications for conservation, global climate change and biomedicine

Seasonal breeding is widespread in vertebrates and involves sequential development of the gonads, onset of breeding activities (e.g. cycling in females) and then termination resulting in regression of the reproductive system. Whereas males generally show complete spermatogenesis prior to and after onset of breeding, females of many vertebrate species show only partial ovarian development and may delay onset of cycling (e.g. estrous), yolk deposition or germinal vesicle breakdown until conditions conducive for ovulation and onset of breeding are favorable. Regulation of this “brake” on the onset of breeding remains relatively unknown, but could have profound implications for conservation efforts and for “mismatches” of breeding in relation to global climate change. Using avian models it is proposed that a brain peptide, gonadotropin-inhibitory hormone (GnIH), may be the brake to prevent onset of breeding in females. Evidence to date suggests that although GnIH may be involved in the regulation of gonadal development and regression, it plays more regulatory roles in the process of final ovarian development leading to ovulation, transitions from sexual to parental behavior and suppression of reproductive function by environmental stress. Accumulating experimental evidence strongly suggests that GnIH inhibits actions of gonadotropin-releasing hormones on behavior (central effects), gonadotropin secretion (central and hypophysiotropic effects), and has direct actions in the gonad to inhibit steroidogenesis. Thus, actual onset of breeding activities leading to ovulation may involve environmental cues releasing an inhibition (brake) on the hypothalamo-pituitary–gonad axis.

In vitro spermatogenesis using bovine testis tissue culture techniques

Spermatogenesis is a complex process initiated by spermatogonial stem cells (SSCs) that have the ability to differentiate into mature spermatozoa or to self-renew to maintain the SSC population and long-term fertility. However, a technique for complete spermatogenesis in vitro using cell culture has not yet been developed. In the present study, we developed in vitro spermatogenesis techniques using bovine testis tissue culture. The effects of specific temperatures and different media on maintaining tubule and germ cell competency were investigated. We found that the optimal temperature and media were 37°C and mouse serum-free medium (mSFM), respectively. In addition, the efficacy of various hormones and growth factors on spermatogenesis in bovine testis tissues maintained in vitro was evaluated. We found that the addition of triiodothyronine (T3) and stem cell factor (SCF) induced spermatogenesis of bovine SSCs in vitro. Therefore, tissue fragments were cultured in the presence of T3 and SCF for three months to induce spermatogenesis in vitro. Overall, in vitro spermatogenesis was enhanced 2.4- to 2.7-fold. Our tissue culture technique may serve as a model system that leads to a more comprehensive understanding of the biology of SSCs as well as the factors that regulate male fertility. Furthermore, the results of this study will be integral for the continued refinement of techniques to manipulate bovine SSCs.

The impact of chronic testicular inflammatory infiltration on spermatogenesis in azoospermic men, evidence-based pilot study

Abstract Introduction: The testes are immune privileged organs but they are subjected to acute and chronic inflammations that can impair spermatogenesis. Spermatogonial stem cells are less sensitive to injury, capable of self-renewal, and act as a reservoir that can restart impaired spermatogenesis. Objective: To study the association of chronic testicular inflammatory infiltration with an area of complete spermatogenesis in testicular biopsies of men with non-obstructive azoospermia (NOA). Design: Retrospective study. Patients and methods: Review of 109 archived Bouin fixed, paraffin embedded and hematoxylin and eosin stained open testicular biopsies of azoospermic men. Main outcome measure: The absence/presence (focal/widespread) of chronic interstitial inflammatory infiltration and absence/presence of late spermatids. Results: Obstructive picture was found in 25 (22.9%) whereas non-obstructive patterns were found in 84 (77.1%) of the reviewed biopsies. Focal interstitial lymphocytic infiltration (ILI) was found in 9 (36%) obstructive biopsies whereas in non-obstructive patterns 32 (38.1%) had focal and 27 (32.1%) had wide spread ILI. In NOA late spermatids were present in 32 (38.1%) biopsies. Finding late spermatids was significantly highest when the infiltrate was focal and significantly lowest when the infiltrate was wide spread. Conclusion: In NOA, the possibility of finding an area of complete focal spermatogenesis in the open testicular biopsy is significantly highest when there is a focal type of ILI, but the possibility is significantly lowest when the infiltrate is widespread. This may guide the clinical testicular sperm extraction (TESE) procedure.

Isoform-Level Gene Expression Profiles of Human Y Chromosome Azoospermia Factor Genes and Their X Chromosome Paralogs in the Testicular Tissue of Non-Obstructive Azoospermia Patients.

The human Y chromosome has an inevitable role in male fertility because it contains many genes critical for spermatogenesis and the development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform-level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with nonobstructive azoospermia (NOA) (40 Sertoli-cell-only syndrome (SCOS) and 28 premiotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level and Western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb, and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1, and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with those of men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm the qPCR results at the protein level, we performed immunoblotting and IHC experiments (based on 24 commercial and homemade antibodies) that detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in azoospermic men. We showed that HSFY1-1, HSFY1-3, BPY2-1, KDM5C2, RBMX2, and DAZL1 transcripts could be used as potential molecular markers to predict the presence of spermatozoa in MA patients. In this study, we have identified isoform level signature that can be used to discriminate effectively between MA, SCOS, and normal testicular tissues and suggests the possibility of diagnosing the presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.