Number Of Testicular Cells Research Articles

Exploring Intercellular Dynamics: Ultra-Weak Biophoton Emission as a Novel Indicator of Altered Cell Functions and Disease in Oligospermia Mice.

Introduction: Biophoton emission, the spontaneous release of photons from living cells, has emerged as an attractive field of research in the study of biological systems. Scientists have recently discovered that changes in biophoton emission could serve as potential indicators of pathological conditions. This intriguing phenomenon suggests that cells might communicate and interact with each other through the exchange of these faint but significant light signals. Therefore, the present study introduces intercellular relationships with biophoton release to detect normal and abnormal cell functions to further achieve cellular interactions by focusing on cell and cell arrangement in disease conditions. Methods: Twenty male mice were assigned to control and busulfan groups. Five weeks after the injection of busulfan, the testis was removed, and then the stereological techniques and TUNEL assay were applied to estimate the histopathology of the testis tissue sections. Results: The findings revealed that the ultra-weak biophoton emission in the control group was significantly lower than in the busulfan group. The oligospermia mice model showed that it significantly changed the spatial arrangement of testicular cells and notably decreased the testis volume, length of seminiferous tubules, and the number of testicular cells. The results of the TUNEL assay showed that the percentage of apoptotic cells significantly increased in the busulfan group. Conclusion: The ultra-weak biophoton emission from testis tissue was reduced in oligospermia mice. As a result, the decline of ultra-weak biophoton can indicate a change in cell arrangement, a decrease in intercellular interaction, and eventually disease.

Photobiomodulation therapy reverses spermatogenesis arrest in hyperthermia-induced azoospermia mouse model.

Testicular heat stress leads to impairment of spermatogenesis in mammals. Involved mechanism in this vulnerability to heat-induced injury remains unclear, and research is being conducted to find an approach to reverse spermatogenesis arrest caused by hyperthermia. Recently, different studies have utilized photobiomodulation therapy (PBMT) therapy for the improvement of sperm criteria and fertility. This study aimed at evaluating the effect of PBMT on the improvement of spermatogenesis in mouse models of hyperthermia-induced azoospermia. A total of 32 male NMRI mice were equally divided into four groups consisting of control, hyperthermia, hyperthermia + Laser 0.03 J/cm2, and hyperthermia + Laser 0.2 J/cm2. To induce scrotal hyperthermia, mice were anesthetized and placed in a hot water bath at 43 °C for 20 min for 5 weeks. Then, PBMT was operated for 21 days using 0.03 J/cm2 and 0.2 J/cm2 laser energy densities in the Laser 0.03 and Laser 0.2 groups, respectively. Results revealed that PBMT with lower intensity (0.03 J/cm2) increased succinate dehydrogenase (SDH) activity and glutathione (GSH)/oxidized glutathione (GSSG) ratio in hyperthermia-induced azoospermia mice. At the same time, low-level PBMT reduced reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation levels in the azoospermia model. These alterations accompanied the restoration of spermatogenesis manifested by the elevated number of testicular cells, increased volume and length of seminiferous tubules, and production of mature spermatozoa. After conducting experiments and analyzing the results, it has been revealed that the use of PBMT at a dosage of 0.03 J/cm2 has shown remarkable healing effects in the heat-induced azoospermia mouse model.

Therapeutic Effects of Edaravone on Azoospermia: Free Radical Scavenging and Autophagy Modulation in Testicular Tissue of Mice.

Background:Chemotherapeutic agents such as cyclophosphamide and busulfan have been shown to have a negative impact on the spermatogenesis process. Based on this fact, the objective of this study was to investigate the effects of edaravone on spermatogenesis in busulfan-induced mice.Methods:Forty adult male mice were equally divided into the four groups: 1) control, 2) edaravone, 3) busulfan, and 4) busulfan + edaravone. Then, the sperm parameters, histopathological examinations, and serum levels of testosterone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were also assessed. Caspase-3, Beclin-1, and ATG-7 mRNA levels were also determined using real-time PCR.Results:Our results revealed that treatment of mice with edaravone in busulfan-induced azoospermia significantly improves sperm parameters, including total count, morphology, and viability (p<0.05). Furthermore, edaravone administration led to a significant increase in serum testosterone (p<0.0001) and FSH (p<0.001) levels, as well as testis weight (p<0.05) and volume (p<0.01). Edaravone also prevented a decrease in the number of testicular cells including spermatogonia (p<0.0001), primary spermatocytes (p<0.001), round spermatids (p<0.0001), Sertoli (p<0.01), and Leydig cells (p<0.0001) in busulfan-treated mice. Additionally, in busulfan-induced azoospermia, edaravone significantly reduced the percentage of sperm with immature chromatin (p<0.0001). Following treatment with edaravone, a decrease in reactive oxygen species (ROS) and an increase in glutathione (GSH) production were noted compared to busulfan-treated mice. Furthermore, caspase-3 (p<0.05), Beclin-1, and ATG-7 (p<0.001) genes expression decreased significantly in treatment groups compared to busulfan-induced azoospermia.Conclusion:According to our findings, edaravone can improve spermatogenesis in busulfan-induced azoospermia through free radical scavenging and autophagy modulation in testicular tissue.

COVID-19 disrupts spermatogenesis through the oxidative stress pathway following induction of apoptosis.

To evaluate the incidence of apoptosis within the testes of patients who died from severe acute respiratory syndrome coronavirus 2 (COVID-19) complications, testis tissue was collected from autopsies of COVID-19 positive (n = 6) and negative men (n = 6). They were then taken for histopathological experiments, and RNA extraction, to examine the expression of angiotensin-converting enzyme 2 (ACE2), transmembrane protease, serine 2 (TMPRSS2), BAX, BCL2 and Caspase3 genes. Reactive oxygen species (ROS) production and glutathione disulfide (GSH) activity were also thoroughly examined. Autopsied testicular specimens of COVID-19 showed that COVID-19 infection significantly decreased the seminiferous tubule length, interstitial tissue and seminiferous tubule volume, as well as the number of testicular cells. An analysis of the results showed that the Johnsen expressed a reduction in the COVID-19 group when compared to the control group. Our data showed that the expression of ACE2, BAX and Caspase3 were remarkably increased as well as a decrease in the expression of BCL2 in COVID-19 cases. Although, no significant difference was found for TMPRSS2. Furthermore, the results signified an increase in the formation of ROS and suppression of the GSH activity as oxidative stress biomarkers. The results of immunohistochemistry and TUNEL assay showed that the expression of ACE2 and the number of apoptotic cells significantly increased in the COVID-19 group. Overall, this study suggests that COVID-19 infection causes spermatogenesis disruption, probably through the oxidative stress pathway and subsequently induces apoptosis.

Involvement of Cytokines and Hormones in the Development of Spermatogenesis In Vitro from Spermatogonial Cells of Cyclophosphamide-Treated Immature Mice.

Aggressive chemotherapy treatment may lead to male infertility. Prepubertal boys do not produce sperm at this age, however, they have spermatogonial stem cells in their testes. Here, we examined the effect of intraperitoneal injection of cyclophosphamide (CP) on the capacity of immature mice (IM) to develop spermatogenesis in vivo and in vitro [using methylcellulose culture system (MCS)]. Our results show a significant decrease in testicular weight, total number of testicular cells, and the number of Sertoli, peritubular, premeiotic, and meiotic/post-meiotic cells, but an increase in the percentages of damaged seminiferous tubules in CP-treated IM compared to control. The functionality of Sertoli cells was significantly affected. The addition of testosterone to isolated cells from seminiferous tubules of CP-treated IM significantly increased the percentages of premeiotic (CD9-positive cells) and meiotic/post-meiotic cells (ACROSIN-positive cells) developed in MCS compared to control. The addition of FSH did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly decreased the percentages of CD9-positive cells and ACROSIN-positive cells. The addition of IL-1 did not affect developed cells in MCS compared to control, but in combination with testosterone, it significantly increased the percentages of VASA-positive cells and BOULE-positive cells compared to IL-1 or testosterone. Addition of TNF significantly increased only CD9-positive cells in MCS compared to control, but in combination with testosterone, it significantly decreased ACROSIN-positive cells compared to testosterone. Our results show a significant impairment of spermatogenesis in the testes of CP-treated IM, and that spermatogonial cells from these mice proliferate and differentiate to meiotic/post-meiotic cells under in vitro culture conditions.

Protective Effect of Quercetin on Testis Structure and Apoptosis Against Lead Acetate Toxicity: an Stereological Study.

Exposure to environmental pollutants tightly impacts on the male fertility. In the present study, we examined the toxic effects of lead acetate (Pb) on testicular structure and the possible effect of quercetin on mitigating these effects. The apoptotic changes in the testes were also studied by the TUNEL assay and changes in apoptosis-related gene (Bax, Bcl-2, and caspase-3) expression. Twenty-one male mice were randomly divided into 3 groups of control, Pb, and lead acetate + quercetin. Testicular weight, both absolute and relative, was higher in Pb-exposed mice in comparison with the control and Pb-quercetin groups. The increase in size of testis was related to the lumen and connective tissue in this group. Lead acetate induced different patterns in testicular cell number; as spermatogonia, spermatocyte, and Sertoli cells number did not affect in lead acetate exposed group, while total number of round spermatids and long spermatids significantly reduced.In addition, Bcl-2 expression was downregulated, and Bax expression was upregulated in Pb-treated group in comparison with the control and Pb + quercetin groups. The TUNEL assay revealed that the number of apoptotic cells in Pb-treated group were increaed significantley in comparison to other groups. In conclusion, Pb administration adversely impacted on the cellular organization and activation of the apoptotic pathways in the testis; on the other hand, quercetin co-administration with lead partially ameliorated these adverse effects.

RETRACTION NOTE TO: The effects of sericin in recovering spermatogenesis and sexual hormone levels in diabetic rats.

Sericin-S (a hydrolysate from the silk protein sericin) is a natural antioxidant, which may improve spermatogenesis while having high biological safety, thus potentially useful for the treatment of male infertility. Our objective was to determine the effects of sericin-S on the sperm parameters and sexual hormone levels in a diabetic rat model. Thirty-six adult male rats were randomly divided in two groups, inducing diabetes in one of them by intraperitoneal administration of streptozotocin (STZ; 65 mg/kg). Both groups were randomly assigned to three subgroups, receiving oral gavage of saline, 1% or 2% sericin extract for 60 days. Therefore, the experimental design was a 2 × 3 factorial design of STZ and sericin treatments. One day after the last treatment, the rats were euthanized, weighed, the testes were processed (weigh, volume, and histology), and serum samples were processed for measuring sex hormone levels [testosterone, follicle-stimulating hormone, and luteinizing hormone (LH)]. STZ treatment decreased LH concentration and counts of spermatogonia, spermatocytes, and spermatids. Body and testis weights were significantly (p < 0.05) lower in the control group (non-diabetic saline) compared to the treated groups. In non-diabetic rats, sericin treatments increased (p < 0.05) the number of testicular cells (spermatogonia, spermatocytes, spermatids, Sertoli, and Leydig cells) and sex hormone concentrations. In diabetic rats, administration of sericin significantly (p < 0.05) improved sperm cell number and sex hormones levels. In nutshell, sericin can clearly modify sperm parameters and overall sex hormone function, and could improve spermatogenesis in normal and diabetic rats.

The effects of sericin in recovering spermatogenesis and sexual hormone levels in diabetic rats.

Sericin-S (a hydrolysate from the silk protein sericin) is a natural antioxidant, which may improve spermatogenesis while having high biological safety, thus potentially useful for the treatment of male infertility. Our objective was to determine the effects of sericin-S on the sperm parameters and sexual hormone levels in a diabetic rat model. Thirty-six adult male rats were randomly divided in two groups, inducing diabetes in one of them by intraperitoneal administration of streptozotocin (STZ; 65mg/kg). Both groups were randomly assigned to three subgroups, receiving oral gavage of saline, 1% or 2% sericin extract for 60days. Therefore, the experimental design was a 2 × 3 factorial design of STZ and sericin treatments. One day after the last treatment, the rats were euthanized, weighed, the testes were processed (weigh, volume, and histology), and serum samples were processed for measuring sex hormone levels [testosterone, follicle-stimulating hormone, and luteinizing hormone (LH)]. STZ treatment decreased LH concentration and counts of spermatogonia, spermatocytes, and spermatids. Body and testis weights were significantly (p < 0.05) lower in the control group (non-diabetic saline) compared to the treated groups. In non-diabetic rats, sericin treatments increased (p < 0.05) the number of testicular cells (spermatogonia, spermatocytes, spermatids, Sertoli, and Leydig cells) and sex hormone concentrations. In diabetic rats, administration of sericin significantly (p < 0.05) improved sperm cell number and sex hormones levels. In nutshell, sericin can clearly modify sperm parameters and overall sex hormone function, and could improve spermatogenesis in normal and diabetic rats.

Sertoli cell-conditioned medium restores spermatogenesis in azoospermic mouse testis.

The current study evaluates potential applications of Sertoli cell (SC)-conditioned medium (CM) and explores the effects of the conditioned medium on the spermatogenesis process in azoospermic mice. For this study, 40 adult mice (28-30g) were divided into 4 experimental groups: (1) control, (2) DMSO 2% (10μl), (3) busulfan (40mg/kg single dose) and (4) busulfan/CM (10μl). SCs were isolated from 4-week-old mouse testes. After using anesthetics, 10μl of CM was injected over 3-5min into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments and RNA extraction in order to examine the expression of c-kit, STRA8 and PCNA genes. The data showed that CM notably increased the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocytes, round spermatids, SCs and Leydig cells compared with the control, DMSO and busulfan groups. Furthermore, the results showed that expression of c-kit and STRA8 was significantly decreased in the busulfan and busulfan/SC groups at 8weeks after the last injection (p < 0.001) but no significant difference was found for PCNA compared with the control and DMSO groups (p < 0.05). These findings suggest that the Sertoli cell-conditioned medium may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine.

Impaired spermatogenesis associated with changes in spatial arrangement of Sertoli and spermatogonial cells following induced diabetes.

The current study was conducted to assess the relationship between testicular cells in spermatogenesis, through which the production of healthy and mature sperm is essential. However, it seems necessary to obtain more information about the three-dimensional pattern of the testis cells arrangement, which is directly related to the function of the testis after induction of diabetes. Twelve adult mice (28-30 g) were assigned into two experimental groups: (1) control and (2) diabetic (40 mg/kg STZ). The epididymal sperm collected from the tail of the epididymis and testes samples were taken for stereology, immunocytochemistry and RNA extraction. Our data showed that diabetes could notably decrease the number of testicular cells, together with a reduction of total sperm count. In addition, the results from the second-order stereology indicated the significant changes in the spatial arrangement of Sertoli cells and spermatogonial cells in the diabetic groups, in comparison with the control (P < .05). Moreover, the immunohistochemistry results showed a significant reduction in Sex-determining Region Y (SRY) box 9 gene (SOX9), vimentin, occludin, and connexin-43 positive cells in the diabetic groups compared with the control (P < .05). Furthermore, our data showed that the expression of steroidogenic acute regulatory protein steroidogenic acute regulatory protein (StAR) and peripheral benzodiazepine receptor peripheral benzodiazepine receptor (PBR) was significantly reduced in the diabetic groups, in comparison with the control (P < .05). These findings suggest that structural and functional changes of testis cells after induction of diabetes cause the alterations in the spatial arrangement of Sertoli and spermatogonial cells, ultimately influencing the normal spermatogenesis in mice.

Effects of Sertoli Cell Transplantation on Spermatogenesis in Azoospermic Mice.

The aim of this study was to evaluate the potential and significant applications of Sertoli cells (SCs) transplantation, and to explore the effect of transplantation on spermatogenesis process, in azospermic mice. In this study, we utilized 18 adult mice (28‒30 g), divided into four experimental groups: (1) control, (2) vehicle (DMSO 2%) (10 µl) (3) busulfan and (4) busulfan+ SCs (1×104 cells/μL). SCs were isolated from the testis of 4-week-old mouse and after using anesthetics, 10 μl of SCs suspension (1×104 cells/μL) was injected over 3-5 min, into each testis and subsequently, sperm samples were collected from the tail of the epididymis. Afterward, the animals were euthanized and testis samples were taken for histopathology experiments, and RNA extraction, in order to examine the expression of c-kit, STRA8 and PCNA genes. Our data showed that SCs transplantation could notably increase the total sperm count and the number of testicular cells, such as spermatogonia, primary spermatocyte, round spermatid, SCs and Leydig cells, compared to the control, DMSO and busulfan groups. Furthermore, the result showed that the expression of c-kit and STRA8 were significantly decreased in busulfan and busulfan/SCs groups, at 8 weeks after the last injection (p<0.001), but no significant decrease was found for PCNA, compared to the control and DMSO groups (P<0.05). These findings suggest that SCs transplantation may be beneficial as a practical approach for therapeutic strategies in reproductive and regenerative medicine. We further highlighted the essential applications that might provide a mechanism for correcting fertility in males, suffering from cell deformity.

Apoptosis, proliferation, and cell size in seasonal changes of body and organ weight in male bank voles Myodes glareolus

Adult male bank voles undergo the body and organ regression before winter, and in early spring, they resume the growth and reproductive processes. The aim of the present study was to determine whether the seasonal changes of body and organ weight in these animals depend on changes in the number of cells or their size. To study an autumnal regression, wild adult males captured in August were exposed to short photoperiod for 0, 4, and 8 weeks, while to study a spring resumption, overwintered males caught in March were exposed to long photoperiod for 0, 1, and 4 weeks. Apoptosis, proliferation, and cell size in the skeletal muscles, liver, and testes were examined. The study revealed that the seasonal changes of testes weight were associated with changes in the number of testicular cells. On the contrary, the changes in size of skeletal myocytes and hepatocytes appeared to be responsible for the seasonal changes of body (muscle) and liver weights in these rodents.

Growth hormone (GH) and GH-releasing hormone (GHRH): Co-localization and action in the chicken testis

Growth hormone (GH) gene expression is not confined to the pituitary gland and occurs in many extrapituitary tissues, including the chicken testis. The regulation and function of GH in extrapituitary tissues is, however, largely unknown. The possibility that chicken testicular GH might be regulated by GH-releasing hormone (GHRH), as in the avian pituitary gland, was investigated in the present study. GHRH co-localized with GH in the germinal epithelium and in interstitial zones within the chicken testes, particularly in the spermatogonia and spermatocytes. In testicular cell cultures, exogenous human GHRH1–44 induced (at 1, 10 and 100nM) a dose-related increase in GH release. Western blot analysis showed a heterogeneous pattern in the GH moieties released during GHRH stimulation. 26kDa monomer GH was the most abundant moiety under basal conditions, but 15 and 17kDa isoforms were more abundant after GHRH stimulation. GHRH treatment also increased the abundance of PCNA (proliferating cell nuclear antigen) immunoreactivity in the testes. This may have been GH-mediated, since exogenous GH similarly increased the incorporation of (3H)-thymidine into cultured testicular cells and increased their metabolic activity, as determined by increased MTT reduction. Furthermore, GH and GHRH immunoneutralization blocked GHRH-stimulated proliferative activity. In summary, these results indicate that GHRH stimulates testicular GH secretion in an autocrine or paracrine manner. Data also demonstrate proliferative actions of GHRH on testicular cell number and suggest that this action is mediated by local GH production.

EFFICIENT AND UNBIASED TOOLS FOR QUANTITATING LEYDIG AND SERTOLI CELLS IN THE TESTIS FROM TESTES BIOPSIES

The possibility of obtaining reliable estimates of the total number of Sertoli and Leydig cells from biopsies by use of the optical fractionator method was tested. The total number of Sertoli and Leydig cells was 522x106 (CV = 0.27) and 89x106 (CV = 0.34), respectively, when sampling in the entire testis while the mean total number of Sertoli and Leydig cells, sampling from biopsies, was 494x106 and 103x106, respectively, statistically a non-significant difference. The present study indicated that total number of testicular cells can be estimated stereologically from the number of cells in biopsies if the sizes of testis and the biopsy can be assessed with adequate precision.

Evaluation of reversible contraceptive activities of Cuminum cyminum in male albino rats

Background The aim of the present study was to evaluate the contraceptive efficacy of Cuminum cyminum (jeera) seeds in male albino rats. Study Design C. cyminum methanol extract (CcMtE) at dose levels of 100 and 200 mg/rat/day was orally administered to male rats for 60 days. The effect of the treatment on reproductive organs and fertility was investigated. Recovery and toxicity studies were also carried out. Results C. cyminum methanol extract fed to male rats for 60 days did not cause any alterations in the body weight, whereas the weight of testes, epididymides, seminal vesicles and ventral prostate were significantly reduced (p≤.001). Animals treated with CcMtE showed a marked reduction in sperm density in the cauda epididymis and testes and sperm motility in the cauda epididymis. Reduction in fertility was 69.0% and 76.0% in 100 and 200 mg/rat/day dose levels, respectively. The circulatory hormones were also reduced significantly. Testicular biochemical analysis of protein, sialic acid, glycogen, ascorbic acid and fructose indicated a marked decline, whereas testicular cholesterol content was significantly increased, which showed altered biochemistry of the reproductive organs. After CcMtE treatment, significant decreases (p≤.001) were observed in the number of testicular cells (i.e., spermatogonia, primary spermatocytes [preleptotene and pachytene], secondary spermatocytes and round spermatids); nonsignificant change was observed in the Sertoli cell count. The treatment had no effect on levels of serum protein, cholesterol, bilirubin, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea and hematological indices. Conclusions The present study shows that C. cyminum treatment resulted in the inhibition of spermatogenesis and fertility without producing apparent toxic effects.

Effect of high intratesticular estrogen on global gene expression and testicular cell number in rats

BackgroundThe identification of estrogen receptors alpha and beta and aromatase in the testis has highlighted the important role of estrogens in regulating spermatogenesis. There is a wealth of information on the deleterious effects of fetal and neonatal exposure of estrogens and xenoestrogens in the testis, including spermiation failure and germ cell apoptosis. However, very little is known about gene transcripts affected by exogenous estradiol exposure in the testis. The objective of the present study was to unveil global gene expression profiles and testicular cell number changes in rats after estradiol treatment.Methods17beta-estradiol was administered to adult male rats at a dose of 100 micrograms/kg body weight in saline daily for 10 days; male rats receiving only saline were used as controls. Microarray analysis was performed to examine global gene expression profiles with or without estradiol treatment. Real time RT-PCR was conducted to verify the microarray data. In silico promoter and estrogen responsive elements (EREs) analysis was carried out for the differentially expressed genes in response to estradiol. Quantitation of testicular cell number based on ploidy was also performed using flow cytometry in rats with or without estradiol treatment.ResultsWe found that 221 genes and expressed sequence tags (ESTs) were differentially expressed in rat testes treated with estradiol compared to the control; the microarray data were confirmed by real time RT-PCR. Gene Ontology analysis revealed that a number of the differentially expressed genes are involved in androgen and xenobiotic metabolism, maintenance of cell cytoskeleton, endocytosis, and germ cell apoptosis. A total of 33 up-regulated genes and 67 down-regulated genes showed the presence of EREs. Flow cytometry showed that estradiol induced a significant decrease in 2n cells (somatic and germ cells) and 4n cells (pachytene spermatocytes) and a marked increase in the number of elongated and elongating spermatids.ConclusionsThis study provides a novel insight into the molecular basis for spermiation failure and apoptosis caused by 17beta-estradiol and it also offers new mechanisms by which adult exposure to environmental estrogens can affect spermatogenesis and fertility.

The effects of dietary protein deficiency on rat testicular function: Der Einfluß einer proteinarmen Diät auf die Hodenfunktion der Ratte

Diets containing 0%, 5% and 10% protein were used for treatment periods of 30, 50, and 90 days respectively. Control rats were fed a diet containing 20% protein. Protein deficient rats failed to gain weight during the experiment. In addition, the weights of the testis, epididymis, prostate and seminal vesicle also decreased, with the 10% group less affected than the 0% and 5% groups. Testicular histology indicated retarded germ cell maturation in the 0% and 5% groups only. Overall testicular cell number and size were reduced in treated rats and there was a reduction in the diameter of the seminiferous tubule in these groups. Epididymal epithelial height was also reduced in protein deficient rats with a concomitant increase in the number of epididymal duct cross sections devoid of sperm. Protein deficiency caused significant reductions in testicular DNA, RNA and protein content. The proportion of motile epididymal sperm decreased in the 0% and 5% groups by 90% and 35% respectively. Epididymal sperm number decreased in both the 0% and 5% groups by 90% while the proportion of abnormal sperm increased by 65% and 61% respectively. Circulating androgen levels were also lowered by more than 50% on average in protein deficient animals.

Dose‐dependent effects on cell proliferation, seminiferous tubules, and male germ cells in the fetal rat testis following exposure to di(n‐butyl) phthalate

Adult male rats gestationally exposed to di(n-butyl)phthalate (DBP) have dysgenetic testes characterized by seminiferous epithelial degeneration, clustering of Leydig cells, and decreased spermatogenesis. Cell proliferation and apoptosis are key processes regulating development of the testis, and alterations in these processes may underlie testicular dysgenesis. To determine whether gestational exposure to DBP affects cell proliferation and apoptosis in the developing rat testis. Pregnant dams were exposed to different dose levels of DBP in mid-gestation and cellular outcomes in fetal and early postnatal testes were assayed by histological and morphometric approaches. Gestational exposure to high dose DBP inhibited proliferation of fetal testicular somatic cells but did not affect apoptosis. Exposed fetal testes had a smaller volume and decreased cell numbers, with decreases in both the tubular and interstitial cell populations. A reduction was observed in the testis volume and altered seminiferous tubule morphometry at > or =50 mg/kg/d, and a decreased testicular cell number at > or =30 mg/kg/d DBP. The number of multinucleated gonocytes in DBP-exposed fetal testes increased after exposure to > or =100 mg/kg/d. The number of proliferating cells in the DBP-exposed testis rapidly rose after birth (when exposure stopped), and the testis volume and the total cell number was comparable to control by postnatal day 2. DBP reversibly inhibits proliferation of somatic cells in the fetal rat testis. Decreased proliferation, rather than increased apoptosis, is the underlying mechanism of altered fetal development of DBP-exposed seminiferous tubules contributing to testicular dysgenesis.

Developmental Changes in Human Fetal Testicular Cell Numbers and Messenger Ribonucleic Acid Levels during the Second Trimester

Normal fetal testis development is essential for masculinization and subsequent adult fertility. The second trimester is a critical period of human testicular development and masculinization, but there is a paucity of reliable developmental data. The objective of the study was to analyze second-trimester human testicular morphology and function. This was an observational study of second-trimester testis development. The study was conducted at the Universities of Glasgow and Aberdeen. Testes were collected from 57 morphologically normal fetuses of women undergoing elective termination of normally progressing pregnancies (11-19 wk gestation). Testicular morphology, cell numbers, and quantitative expression of 22 key testicular genes were determined. Sertoli cell and germ cell number increased exponentially throughout the second trimester. Leydig cell number initially increased exponentially but slowed toward 19 wk. Transcripts encoding Sertoli (KITL, FGF9, SOX9, FSHR, WT1) and germ (CKIT, TFAP2C) cell-specific products increased per testis through the second trimester, but expression per cell was static apart from TFAP2C, which declined. Leydig cell transcripts (HSD17B3, CYP11A1, PTC1, CYP17, LHR, INSL3) also remained static per cell. Testicular expression of adrenal transcripts MC2R, CYP11B1, and CYP21 was detectable but unchanged. Expression of other transcripts known or postulated to be involved in testicular development (GATA4, GATA6, CXORF6, WNT2B, WNT4, WNT5A) increased significantly per testis during the second trimester. The second trimester is essential for the establishment of Sertoli and germ cell numbers. Sertoli and Leydig cells are active throughout the period, but there is no evidence of changing transcript levels.

The effects of oestrogen receptors α and β on testicular cell number and steroidogenesis in mice

Oestrogen plays an important role in testicular function. This study used mice null for oestrogen receptor alpha (ER alpha) or beta (ER beta) to investigate which receptor mediates the effects of oestrogen within the testis. Groups of ER alpha knockout mice (alpha ERKO) and ER beta knockout mice (beta ERKO) and wild-type littermates (n=5-8) were killed at 11 weeks post partum. One testis was fixed in Bouin's fluid for stereology and the other frozen for testosterone measurement. Trunk blood was collected for testosterone RIA. The optical disector combined with the fractionator methodology was used to estimate Leydig, Sertoli and germ cell numbers. At all times, the knockout animals were compared with their wild-type littermates. The physical disector quantified cells stained immunohistochemically for the apoptotic marker active caspase-3 and Hoechst staining was used to identify nuclear fragmentation. The mean Leydig cell volume was measured using the point sampled intercept method. The Leydig cell number per testis was significantly increased in beta ERKO mice but not in alpha ERKO mice. Plasma and testicular testosterone concentrations were increased in alpha ERKO mice but no changes were observed in beta ERKO mice. Hypertrophic Leydig cell changes were observed in alpha ERKO mice, and a decreased mean cell volume was seen in beta ERKO mice. No difference in Sertoli cell number per testis was observed in any of the groups. The spermatogonial cell number per testis was increased in beta ERKO mice. Immunohistochemistry identified increased numbers of active caspase-3-labelled germ cells per testis in alpha ERKO mice but not beta ERKO mice. Hoechst staining supported these findings. There was significant germ cell loss in alpha ERKO mice. This study suggests that ER beta may be involved in regulation of Leydig cell proliferation and testosterone production in the adult mouse testis.