• Anatomy and Cell Biology
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• v.56 no.4
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• pp.526-537
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• 2023

Hepatitis C virus (HCV) infection is a major health problem worldwide and its eradication is mandatory. Direct acting HCV polymerase inhibitors, such as Sofosbuvir (SOF), is an effective regimen. However, it has some side effects like mutagenesis, carcinogenesis, and the impairment of testicular function. It is important to evaluate the safety of SOF on the ovary, as there are no studies yet. Increasing the production of Reactive Oxygen Species (ROS), causes oxidative stress, which affects ovulation process, female reproduction, and fertility. Accumulation of SOF in the cells was demonstrated to promote ROS generation. Vitamin E (Vit E) is an antioxidant agent that has an essential role in the female reproductive system, its deficiency can cause infertility. We explored the effect of SOF treatment alone and co-treated with Vit E on ovarian ROS level and ovarian morphology experimentally using biochemical and immunohistochemical studies. Significant changes in oxidative stress markers; nitric oxide and malondialdehyde lipid peroxidation, antioxidant enzymes; catalase, super oxide dismutase, and reduced glutathione, proliferating markers; proliferation cell nuclear antigen and Ki-67 antigen and caspase 3 apoptotic marker were demonstrated. It was shown that where SOF induced oxidative stress, it also aggravated ovarian dysfunction. The essential role of Vit E as an antioxidant agent in protecting the ovarian tissue from the effect of oxidative stress markers and preserving its function was also displayed. This could be guidance to add Vit E supplements to SOF regimens to limit its injurious effect on ovarian function.

• Anatomy and Cell Biology
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• v.56 no.4
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• pp.508-517
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• 2023

In cancer patients, chemo/radio therapy may cause infertility by damaging the spermatogenesis affecting the self-renewal and differentiation of spermatogonial stem cells (SSCs). In vitro differentiation of stem cells especially mesenchymal stem cells (MSCs) into germ cells has recently been proposed as a new strategy for infertility treatment. The aim of this study was to evaluate the proliferation and differentiation of SSCs using their co-culture with Sertoli cells and conditioned medium (CM) from adipose tissue-derived MSCs (AD-MSCs). Testicular tissues were separated from 2-7 days old neonate Wistar Rats and after mechanical and enzymatic digestion, the SSCs and Sertoli cells were isolated and cultured in Dulbecco's modified eagle medium with 10% fetal bovine serum, 1X antibiotic, basic fibroblast growth factor, and glial cell line-derived neurotrophic factor. The cells were treated with the CM from AD-MSCs for 12 days and then the expression level of differentiation-related genes were measured. Also, the expression level of two major spermatogenic markers of DAZL and DDX4 was calculated. Scp3, Dazl, and Prm1 were significantly increased after treatment compared to the control group, whereas no significant difference was observed in Stra8 expression. The immunocytochemistry images showed that DAZL and DDX4 were positive in experimental group comparing with control. Also, western blotting revealed that both DAZL and DDX4 had higher expression in the treated group than the control group, however, no significant difference was observed. In this study, we concluded that the CM obtained from AD-MSCs can be considered as a suitable biological material to induce the differentiation in SSCs.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.4
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• pp.253-261
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• 2023

Objective: Azoospermia (the total absence of sperm in the ejaculate) affects approximately 10% of infertile males. Despite diagnostic advances, azoospermia remains the most challenging issue associated with infertility treatment. Our study evaluated transition nuclear protein 2 (TNP2) and synaptonemal complex protein 3 (SYCP3) polymorphisms, azoospermia factor a (AZFa) microdeletion, and gene expression levels in 100 patients with azoospermia. Methods: We investigated a TNP2 single-nucleotide polymorphism through polymerase chain reaction (PCR) restriction fragment length polymorphism analysis using a particular endonuclease. An allele-specific PCR assay for SYCP3 was performed utilizing two forward primers and a common reverse primer in two PCR reactions. Based on the European Academy of Andrology guidelines, AZFa microdeletions were evaluated by multiplex PCR. TNP2, SYCP3, and the AZFa region main gene (DEAD-box helicase 3 and Y-linked [DDX3Y]) expression levels were assessed via quantitative PCR, and receiver operating characteristic curve analysis was used to determine the diagnostic capability of these genes. Results: The TNP2 genotyping and allelic frequency in infertile males did not differ significantly from fertile volunteers. In participants with azoospermia, the allelic frequency of the SYCP3 mutant allele (C allele) was significantly altered. Deletion of sY84 and sY86 was discovered in patients with azoospermia and oligozoospermia. Moreover, SYCP3 and DDX3Y showed decreased expression levels in the azoospermia group, and they exhibited potential as biomarkers for diagnosing azoospermia (area under the curve, 0.722 and 0.720, respectively). Conclusion: These results suggest that reduced SYCP3 and DDX3Y mRNA expression profiles in testicular tissue are associated with a higher likelihood of retrieving spermatozoa in individuals with azoospermia. The homozygous genotype TT of the SYCP3 polymorphism was significantly associated with azoospermia.

• Development and Reproduction
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• v.27 no.3
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• pp.149-157
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• 2023

We investigated the involvement of autophagy with steroidogenesis in testicular Leydig cells. Human chorionic gonadotropin (hCG)-stimulated T production in Leydig cells was not remarkably altered in the presence of an autophagy inhibitor 3-methyladenine (3-MA). Although pretreatment with 3-MA demonstrated a tendency to decrease hCG-induced T production, the differences were significant only at a higher time point of 24 h following hCG. Microtubule associated protein light chain 3 (LC3)-II was detectable in the control cells in all the experiments. The hCG-induced increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleave (P450scc) protein levels were not significantly altered by 3-MA. Leydig cells isolated from immature rat testes 12 h following hCG treatment showed relatively increased levels of LC3-II protein compared to the control group. Furthermore, LC3-II levels shown in these cells reached almost the identical to those from normal adult testes. However, LC3-II protein levels were almost comparable or even slightly lower than the controls at 48 h following hCG. Expression of StAR and P450scc was upregulated at both 12 and 48 h after hCG. We also used MA-10 cells, the mouse Leydig cell line, in this experiment. When dibutyryl cyclic-AMP was treated with MA-10 cells, P4 levels were significantly increased in the cell culture medium. However, P4 levels tended to decrease in the presence of 3-MA, but the difference was not statistically significant. This was consistent with the results of the rat Leydig cell experiments. Together, we believe that although autophagy participates in steroidogenesis and enhances steroidogenic efficacy of Leydig cells, it may not be a decisive cellular process for steroidogenesis, specifically in the mature Leydig cells.

• Development and Reproduction
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• v.27 no.4
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• pp.175-183
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• 2023

The epididymal fat is a type of gonadal adipose tissue, which is localized closely to the testis. Even though it has been suggested that the epididymal fat is necessary for maintenance of spermatogenesis in the testis, the influence of epididymal fat on expression of testicular steroidogenic enzymes has not been examined. In the present research, expressional changes of steroidogenic enzymes in the mouse testis after 2 weeks of the surgical partial lipectomy of epididymal fat at different postnatal ages were determined by real-time polymerase chain reaction analysis. The transcript levels of all molecules at 2 months of postnatal age were significantly increased by the lipectomy of epididymal fat. However, the lipectomy at 5 months of postnatal age resulted in decreases of expression levels of all molecules examined in the testis. Except a reduced transcript level of hydroxysteroid 17-beta dehydrogenase 3, there were no significant changes of expression levels of other steroidogenic enzymes by the lipectomy at 8 months of postnatal age. At 12 months of postnatal age, the lipectomy caused a significant increase of transcript level of steroidogenic acute regulatory protein and a significant decrease of transcript level of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1, without any expressional change of cytochrome P450 side chain cleavage, hydroxysteroid 17-beta dehydrogenase 3, and hydroxysteroid 17-beta dehydrogenase 3 in the testis. These findings suggest that the substances derived from epididymal fat could differentially influence on expression of steroidogenic enzymes in the testis during postnatal period.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.253-260
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• 2004

Objectives: Klinefelter syndrome is the most common genetic cause of male infertility and presents with 47, XXY mainly or 46, XX/47, XXY mosaicism. It is characterized by hypogonadism and azoospermia due to testicular failure, however, sporadic cases of natural pregnancies have been reported. With the development of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI), sperm can be retrieved successfully and ART is applied in these patients for pregnancy. It has been suggested that the risk of chromosome aneuploidy for both sex chromosome and autosome is increased in the sperms from 47, XXY germ cells. Considering the risk for chromosomal aneuploidy in the offspring, preimplantation genetic diagnosis (PGD) could be applied as a safe and more effective treatment option in Klinefelter syndrome. The aim of this study is to assess the outcome of PGD cycles by using FISH for sex chromosome and autosome in patients with Klinefelter syndrome. Materials and Methods: From Jan. 2001 to Dec. 2003, PGD was attempted in 8 cases of Klinefelter syndrome but TESE was failed to retrieve sperm in the 3 cases, therefore PGD was performed in 8 cycles of 5 cases (four 47, XXY and one 46, XY/47, XXY mosaicism). In one case, ejaculated sperm was used and in 4 cases, TESE sperm was used for ICSI. After fertilization, blastomere biopsy was performed in $6{\sim}7$ cell stage embryo and the chromosome aneuploidy was diagnosed by using FISH with CEP probes for chromosome X, Y and 17 or 18. Results: A total of 127 oocytes were retrieved and ICSI was performed in 113 mature oocytes. The fertilization rate was $65.3{\pm}6.0%$ (mean$\pm$SEM) and 76 embryos were obtained. Blastomere biopsy was performed in 61 developing embryos and FISH analysis was successful in 95.1% of the biopsied blastomeres (58/61). The rate of balanced embryos for chromosome X, Y and 17 or 18 was $39.7{\pm}6.9%$. The rate of aneuploidy for sex chromosome (X and Y) was $45.9{\pm}5.3%$ and $43.2{\pm}5.8%$ for chromosome 17 or 18, respectively. Embryo transfer was performed in all 8 cycles and mean number of transferred embryos was $2.5{\pm}0.5$. In 2 cases, clinical pregnancies were obtained and normal 46, XX and 46, XY karyotypes were confirmed by amniocentesis, respectively. Healthy male and female babies were delivered uneventfully at term. Conclusion: The patients with Klinefelter syndrome can benefit from ART with TESE and ICSI. Considering the risk of aneuploidy for both sex chromosome and autosome in the sperms and embryos of Klinefelter syndrome, PGD could be offered as safe and more effective treatment option.

• Korean Journal of Poultry Science
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• v.33 no.3
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• pp.181-188
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• 2006

Changes in the fine structure of testicular Leydig cell from hatching to adulthood were studied in Korean native chickens of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 21, 24, 28, 32, 44, 52 and 64 weeks (n=13 chickens per group) of age. The objective of this study were to elucidate Leydig cell ultrastructure during testicular development. Testes of chickens were fixed by whole body perfusion using a fixative containing 2.5% glutaraldehyde in cacodylate buffer, processed and embedded in Epon-araldite. Using $1{\mu}m$ sections stained with methylene blue-azure II, qualitative and quantitative(stereological) morphological studies were performed. The ultrastructural changes of the Leydig cell were investigated by ultrathin section with the transmission electron microscope. The stages of the Leydig cell development described focus on mitochondria, endoplasmic reticulum, and lipid droplets which are involved in androgens as fullows. 1) Approaching puberty. The closely packed Leydig cells and sparse intercellular space. The nucleus occupied a large portion of the Leydig cell volume. The population of Leydig cells contained two types of cells that differed in the appearance of their nuclei which were either highly electron-opaque or relatively electron-lucid. The cytoplasm was characterized by large amounts of lipid droplets, relatively few spherical mitochondria, and sparse smooth endoplasmic reticulum. 2) Puberty to adult. The Leydig cells which display features compatible with significant androgen synthesis: large volume of cytoplasm containing extended smooth endoplasmic reticulum, abundant mitochondria, and reduction of lipid droplets.

• Development and Reproduction
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• v.14 no.4
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• pp.281-286
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• 2010

Recent evidence has revealed that the intratesticular injection of hypertonic saline(20%) resulted in a chemically castrated state such as nadir testosterone levels in rats. To confirm the efficacy of this simple saline-injection method further, we investigated the changes in the gross and microscopic anatomy of testis. Our study comprised three groups; intact(control) group, orchidectomy group and saline-injection (experimental) group. Single dose of hypertonic saline (sterilized, $750{\mu}{\ell}/testis$) were directly administered into both testis of adult rats (about 300 g BW). Bilateral orchidectomy was performed at the same day of saline injection. Following 30 days post-injection, reproductive tissues were surgically removed, weighed and fixed for histological examination. The body weights were not changed in both orchidectomy group and saline-injection group when compared to those in intact group. The wet weights of testis were significantly decreased in saline-injection group when compared to those in intact group. The wet weights of epididymis and seminal vesicle and prostate were significantly decreased in orchidectomy group and saline-injection group when compared to those in intact group. Macroscopically, the testes exerted slight atrophy and the tunica albuginea seemed to be intact in saline injection group. Histologically, however, larger parts of testicular tissue underwent necrosis and were barely recognizable after hematoxylin-eosin staining. In the same section, only the opposite part of the injection site was stained showing abnormal state of cell layers mostly fibrosis and infiltrated leukocytes. Sloughing of immature germ cells from the basement membrane along with shedding cells in the intraluminal space was notable in most seminiferous tubules from the saline injected testis. The present study confirmed that the direct injection of hypertonic saline into testis can induce a castration-like, testosterone-depriving effects on accessory sex organs. Our findings suggest that the efficacy of this less expensive and minimally invasive method seems to be almost even with that of conventional orchidectomy and chemical castration, though more in-depth evaluation should be supported.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.13 no.4
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• pp.125-134
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• 1980

The dovelopment of the gonads, gametogenesis and the reproductive cycle of the topshell, Turbo cornutus Solander, which is one of valuable food animals fom Korean waters were studied by photomicroscophy. The materials were monthly collected from Bangeojin, Jeongjari and Dangweol, all these places being located in the south-eastern part of Korea, for one year from March 1979 to February 1980. Topshell is dioecious and oviparous. Gonad is situated on the surface of liver, which lies posteriorly. The surface of ovary and testis is covered with a fibrous membrane, membrane of connective and muscular fibers and then an outermost layer of simple-columnar epithelial cells which are composed of cuboidal and columnar mucous gland cells. Primordial germ cells develop on the germinal epithelium of ovarian and testicular lobuli which are originated from the fibrous membrane and extend toward hepatic gland. Undifferentiated mesenchymal tissue and pigment granular cells are abundantly distributed between the growing oocytes and spermatocytes in the early development stages. With the further development of the ovary and testis these tissue and cells gradually disappear. Then the undifferentiated mesenchymal tissue and pigment granular cells are considered to be related to the growing of the oocytes and spermatocytes. Early multiplicating oogonium is ca. $10\mu$ in diameter and nucleushaving a central nucleolus is ra. $8\mu$. As the oocytea grow to ca. $50-60\mu$ by the increase of cytoplasm, the oocytes become look like bunches of grapes which are attached to ovarian lobuli. Mature eggs are ca. $180-210\mu$ in diameter and it is surrounded by a gelatinous membrane of ca. $10\mu$ in thickness. After spawning, undischarged ripe eggs and spermatozoa remain in the ovary and testis respectively for some time. Then they finally degenerate, and proliferation of new oogonia and spermatogonia occur along the germinal epithelia of newly developed ovarian and testicular lobuli. Reprocuctive cycle of Turbo cornutus could be classified into five successive stages: multiplicative, growing, maturer spent and recovery stages. Spawning occurs from August to November with Peak spawning from early September to late October.

• Korean Journal of Ichthyology
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• v.18 no.4
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• pp.311-318
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• 2006

Ultrastructure of germ cells, the cyst epithelial cells and interstitial cells during spermatogenesis of the stone flounder, Kareius bicoloratus (Pleuronectidae) sampled on the west coast of Korea were investigated by electron microscopic observations. In the primary spermatocyte, the synaptonemal complexes appear in the zygotene stage of the prophase during maturation division. In the growing testis, especially, the interstitial cells (Leydig cells) appear near the primary, secondary spermatocytes and spermatids. Well-developed interstitial cells (steroid hormone secreting cells) which are located in the interlobular space in growing testis have three morphological characteristics of a vesicular nucleus, mitochondria with tubular cristae and smooth endoplasmic reticulum. During spermatogenesis, the primary and secondary spermatocytes attach to the cyst epithelial cell (Sertoli cell) having an elongated ovoid or triangular nucleus and several mitochondria in the cytoplasm. In the growing testis, lipid droplets, the mitochondrial rosettes and glycogen particles appear in the cytoplasm of the cyst epithelial cells near the secondary spermatocytes and spermatids. Particularly, the mitochondria, endoplasmic reticulum, little lipid droplets and the large amount of glycogen particles are present in the cytoplasm of the cyst epithelial cell in the late growing testis. In the late stage of spermiogenesis, the proximal centriole is joined to the nuclear envelope, the distal centriole forms the basal body of the flagellum and gives rise to the axial filament of the flagellum. No acrosome of the sperm is formed as seen in other teleost fish. The head of the spermatozoon is approximately $3{\mu}m$ in length and its tail is about $30{\mu}m$ in length. The axoneme of the tail flagellum of the spermatozoon consists of nine outer doublet microtubules at the periphery and two centrial singlet microtubules at the center. The spermatozoon of this species has two axonemal lateral fins. Especially, the cyst epithelial cells which located near groups of gametes in the various stages, show three functions: nutrition, phagocytosis and steroidogenesis. Especially, the nuclei of cyst epithelial cells in the recovery stage of the testicular developmental stages appear to be irregular in shape after spermiation. Of three functions of the cyst epithelial cell, several characteristics of phagocytosis are showed in the cytoplasm of the cyst epithelial cells in the recovery stage of the testicular developmental stages. At this stage, therefore, it is assumed that the cyst epithelial cells are involved in degeneration and resorption of undischarged germ cells after spermiation.