Immature Rat Testis Research Articles

The long noncoding RNA (LINC-RBE) expression in testicular cells is associated with aging of the rat.

Long noncoding RNAs (lncRNAs) are important regulatory biomolecules responsible for many cellular processes. The aging of mammals is manifested by a slow and gradual decline of physiological functions after adulthood, progressively resulting in age-related diseases. Testis comprises different cell-types with defined functions for producing haploid gametes and androgens in males, contributing gene-pool to the next generation with genetic variations to species for evolutionary advantage. The LINC-RBE (long intergenic noncoding-rat brain expressed) RNA showed highest expression in the Leydig cells, responsible for steroidogenesis and production of testosterone; higher expression in primary spermatocytes (pachytene cells), responsible for generation of haploid gametes and high expression in Sertoli cells, the nursing cells of the testes. Testes of immature (4-weeks), adult (16- and 44-weeks), and nearly-old (70-weeks) rats showed low, high, and again low levels of expression, respectively. This along with the nuclear-cytoplasmic localization of LINC-RBE RNA showed age-related expression and function. Thus, expression of LINC-RBE is involved in the molecular physiology of testes, especially Leydig cells, primary spermatocytes, and Sertoli cells. The decline in its expression correlates with diminishing reproductive function of the testes during aging of the rat.

X-box binding protein 1 caused an imbalance in pyroptosis and mitophagy in immature rats with di-(2-ethylhexyl) phthalate-induced testis toxicity

As a widely used plasticizer, di-(2-ethylhexyl) phthalate (DEHP) is known to induce significant testicular injury. However, the potential mechanism and effects of pubertal exposure to DEHP on testis development remain unclear. In vivo, postnatal day (PND) 21 male rats were gavaged with 0, 250, and 500 mg/kg DEHP for ten days. Damage to the seminiferous epithelium and disturbed spermatogenesis were observed after DEHP exposure. Meanwhile, oxidative stress-induced injury and pyroptosis were activated. Both endoplasmic reticulum (ER) stress and mitophagy were involved in this process. Monoethylhexyl phthalate (MEHP) was used as the biometabolite of DEHP in vitro. The GC-1 and GC-2 cell lines were exposed to 0, 100 μM, 200 μM, and 400 μM MEHP for 24 h. Reactive oxygen species (ROS) generation, oxidative stress damage, ER stress, mitophagy, and pyroptosis were significantly increased after MEHP exposure. The ultrastructure of the ER and mitochondria was destroyed. X-box binding protein 1 (XBP1) was observed to be activated and translocated into the nucleus. ROS generation was inhibited by acetylcysteine. The levels of antioxidative stress, ER stress, mitophagy, and pyroptosis were decreased as well. After the administration of the ER stress inhibitor 4-phenyl-butyric acid, both mitophagy and pyroptosis were inhibited. Toyocamycin-induced XBP1 down-regulation decreased the levels of mitophagy and pyroptosis. The equilibrium between pyroptosis and mitophagy was disturbed by XBP1 accumulation. In summary, our findings confirmed that DEHP induced a ROS-mediated imbalance in pyroptosis and mitophagy in immature rat testes via XBP1. Moreover, XBP1 might be the key target in DEHP-related testis dysfunction.

Immature rat testis sustained long-term development using an integrative model

BackgroundXenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model.MethodsIn the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis.Results(a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05).ConclusionThe new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.

Age features of morphological changes of vessels of testicular hemomicrocirculatory flow in streptozotocin diabetes mellitus

Medico-social problem of diabetes is caused by early disability and mortality of patients due to specific complications of micro- and macroangiopathies. Therefore, the aim of our study was to establish morphological changes in vessels of the hemomicrocirculatory flow of the testes of immature rats with experimental streptozotocin diabetes mellitus (SDM). The material for the study were the testicles of 20 two-month-old immature (weighing 65-95 g) white outbred male rats, which were divided equally into 2 groups: experimental and control ones. SDM in animals of the experimental group was simulated by a single intraperitoneal injection of streptozotocin (dissolved in 0.1 M citrate buffer solution with a pH of 4.5) at a dose of 7 mg per 100 g of mass. The control group of animals received intraperitoneally an equivalent dose of 0.1 M citrate buffer. Histological, electron microscopic, biochemical, morphometric and statistical research methods were used.&#x0D; It was found that in the early stages of SDM (14th day) on the background of hyperglycemia in the hemomicrocirculatory flow of the testes there is a spasm of the vessels of the afferent link, which is confirmed by a decrease in the area of arterioles lumen and an increase in their VI.&#x0D; On the 56th day of SDM, on the background of elevated levels of glucose and glycosylated hemoglobin in the links of the hemorrhagic circulatory flow of the testes there are initial signs of diabetic microangiopathy, manifested by: hemorheological disorders in micro-hemo-vessels (erythrocyte sludges, adhesion of erythrocytes and platelets, microclasmatosis), decreased capacity of arterioles and capillaries (increase in VI, respectively by 1.2 and 1.9 times), microclasmatosis, thickening and proliferation of the basement membrane of capillaries.&#x0D; Thus, on the 56th day of SDM in the hemomicrocirculatory flow of the testes, the development of diabetic microangiopathy is observed, which leads to the disruption of the blood-testis barrier, and as a consequence, to a violation of spermatogenesis.

Estradiol and 1α,25(OH)2 vitamin D3 share plasma membrane downstream signal transduction through calcium influx and genomic activation in immature rat testis

The aim of this study was to investigate the rapid response pathway and gene and protein expression profiles of the rat testis in response to estradiol (E2) and 1α,25(OH)2 vitamin D3 (1,25-D3), to understand how they mediate their effects on the first spermatogenic wave. To do this, we compared the effects of 1,25-D3 and E2 on 45calcium(Ca2+) uptake and the involvement of estrogen receptors (ESR) in their rapid responses. Additionally, we studied the downstream signal transduction effects of 1,25-D3 and E2 on cyclin A1/B1 and cellular cycle protein expression. As previously observed for 1,25-D3, E2 also increased 45Ca2+ uptake in immature rat testes via voltage-dependent Ca2+ channels, Ca2+-dependent chloride channels and via the activation of protein kinase C, protein kinase A and mitogen-activated protein kinase kinase (MEK). Elevated aromatase expression by testes was observed in the presence of 1,25-D3 and both hormones decreased ESR mRNA expression. Furthermore, 1,25-D3 and E2 diminished cyclin A1 mRNA expression, but E2 did not affect cyclin B1 mRNA levels. Consistent with these findings, the immunocontent of cyclin A1 and B1 in the testes was also increased by 1,25-D3 and E2. 1,25-D3 increased expressions of the p16 and p53 proteins, supporting the anti-proliferative and pro-apoptotic properties of 1,25-D3, while E2 also augmented p16. Data indicate that both hormones trigger rapid responses at the plasma membrane that may control the expression of gene and proteins related to cell cycle regulation, and thereby modulate spermatogenesis.

The Use of Fullerene C60 to Preserve Testicular Tissue after Cryopreservation

Autologous transplantation of cryopreserved fragments of an immature testis is an actively developing approach to save fertility in patients facing a gonadotoxic therapy. The use of bioavailable fullerene C60 as a powerful antioxidant opens up a new potential for the prevention and correction of ischemic-reperfusion pathological processes in tissues including those associated with freezing-thawing procedure. In this work, we aimed to study the antioxidant status, apoptotic/necrotic processes, and morphological characteristics of cryopreserved fragments of the seminiferous tubules of testis (CrFSTT) of immature rats after incubation in media with different concentrations of fullerene C60 (10, 15, and 20 μg/mL). Our results indicated that the addition of C60 in a concentration of 15 μg/mL decreased ROS production, cytochrom C release, and degree of histological damage of spermatogenic epithelium as well as increased the activity of the mitochondria, antioxidant defense system, and cell density in histological sections of CrFSTT compared to the control. Fullerene C60 at investigated concentrations did not impact significantly on apoptosis in cells of CrFSTT but, after incubation with 15 μg/mL C60, a percentage of living cells was 1.2-fold higher and a value of necrotic ones in this group was 1.6-fold lower than the control samples ( p &lt; 0.05 ). Relative amount of cells of the spermatogonia germ layer did not differ between the studied concentrations. The general analysis of obtained data showed that the C60 addition in the concentration of 15 μg/mL was the most optimal for the rehabilitation of CrFSTT. The results can be used for the development of an effective rehabilitation medium for the cryopreserved testicular tissue.

APPLICATION EFFICIENCY OF BOVINE SERUM ALBUMIN FOR RECOVERY OF SEMINIFEROUS TUBULES OF TESTES AFTER CRYOPRESERVATION

An optimal approach to the recovery of testicular tissue fragments after cryopreservation is critical for their further use in order to successful fertility restoration. Aim. The purpose of this study was to investigate the effect of bovine serum albumin (BSA) addition to the rehabilitation medium on the morphofunctional characteristics of fragments of seminiferous tubules of testes (FSTT) of immature rats after cryopreservation. Methods. The object of the study was cryopreserved by slow cooling and vitrified FSTT. Warmed samples were incubated for 30 min in Leibovitz medium supplemented with BSA at concentrations of 0, 2, 5 or 10%. After that, morphological characteristics, the activity of the metabolic and antioxidant systems were evaluated. Results. It was found that in the samples cryopreserved by slow cooling, the use of 5% BSA contributed to the increase in the safety of spermatogenic epithelium cells, in the levels of metabolic and antioxidant activities. In case of vitrified FSTT samples, it was observed that the addition of 5% BSA to the medium promoted the repair of minor tissue damage and increased metabolic activity level, but did not affect the state of the antioxidant defense system. Conclusions. The obtained data can be used to develop an effective rehabilitation medium for cryopreserved fragments of the seminiferous tubules of testes using BSA.

A Brazilian pulp and paper mill effluent disrupts energy metabolism in immature rat testis and alters Sertoli cell secretion and mitochondrial activity.

Our objective was to investigate whether the pulp and paper mill industry effluent could affect the testis and Sertoli cells in a fast exposure period. For this, the present study was carried out in immature rats at 10-day-old. Testis treated in vitro with 4% effluent for 1 h presented changes in energy metabolism in terms of a decrease in lactate content and glucose uptake. Elevation in GSH content, as an antioxidant defense mechanism, was also detected. Sertoli cells treated with 4% effluent for 1 hour showed alterations in the mitochondrial metabolism that favor the decoupling of oxidative phosphorylation and the generation of oxygen reactive species and also a time and concentration-dependent delay secretion of acidic vesicles. Our results showed that pollutants present in the pulp and paper mill effluents, in a short time of exposure, are capable of inducing alterations in important metabolic functions in the testis and in Sertoli cells that are crucial for the correct progression of spermatogenesis and fertility.

Crosstalk in the non-classical signal transduction of testosterone and retinol in immature rat testes

This study aimed to investigate the effects of the interaction between testosterone and retinol on the rapid responses of cultured Sertoli cells obtained from 10-day-old immature rat testes. Non-classical actions of testosterone and retinol were investigated, and the activities of L-type voltage-dependent calcium channels (L-VDCC) and voltage-dependent potassium channels (Kv) were determined by measuring 45Ca2+ influx in whole testis. Additionally, the effects of testosterone and retinol on these channels were studied in primary culture of Sertoli cells using the patch-clamp technique. 45Ca2+ influx was used to observe a dose–response curve on tissues treated with retinol and/or testosterone for 2 min (10−12, 10−9 and 10−6 M and 10−9 and 10−6 M), and a concentration of 10−6 M was selected to investigate the mechanism of action of testosterone and retinol on rapid responses. Participation of the L-VDCC and Kv channels was investigated using nifedipine and tetraethylammonium chloride (TEA) inhibitors, respectively. Both, testosterone and retinol act through non-classical mechanisms, stimulating 45Ca2+ influx in immature rat testes. The response to testosterone was abolished by nifedipine and TEA, whereas the effects of retinol were partially blocked by nifedipine and completely inhibited by TEA. Retinol amplified the testosterone-induced effect on 45Ca2+ influx in the testes, suggesting a crosstalk between rapid responses (calcium influx) and cell repolarization via activation of Kv channels. Whole-cell electrophysiology data demonstrated that testosterone and retinol increased voltage-dependent potassium currents (Kv) in Sertoli cells; inhibition of these responses by TEA confirmed the involvement of TEA-sensitive K+ channels in these effects. Taken together, we demonstrate, for the first time, crosstalk between testosterone and retinol that is mediated by a non-classical mechanism involving the L-VDCC-triggered cell depolarization and activation of repolarization by Kv currents in Sertoli cells. These ionic modulations play a physiological role in Sertoli cells and male fertility via stimulation of secretory activities.

Altered hormonal milieu and dysregulated protein expression can cause spermatogenic arrest in ectopic xenografted immature rat testis

Testis tissue xenografting complemented with cryopreservation is a feasible technique for fertility preservation in children with malignancy receiving gonadotoxic therapy and for endangered species with high neonatal mortality rate. However, xenografted testis of human and most endangered species are known to undergo spermatogenic arrest. In this study, we xenografted immature rat testis onto immunodeficient male mice to investigate the plausible underlying causes of spermatogenic arrest. Histological analysis of xenografted testes collected 8-wk post-grafting showed incomplete spermatogenesis with pachytene-stage spermatocytes as the most advanced germ cells. Although the levels of serum luteinizing hormone and testosterone were normal in recipient mice, those of follicle stimulating hormone (FSH) were significantly high, and specific receptors of FSH were absent in the xenografts. The xenografts demonstrated dysregulated expression of Sertoli cell-transcriptional regulators (WT1 and SOX9) and secretory proteins (SCF and GDNF). In conclusion, results from our study suggested that an altered hormonal milieu in recipients and dysregulated protein expression in xenografts could be a potential cause of spermatogenic arrest in xenografted immature rat testis. Further stereological analysis of xenografts can demonstrate precise cellular composition of xenografts to decipher interactions between germ and somatic cells to better understand spermatogenic arrest in xenografted testis.

Drp1 Phosphorylation Is Indispensable for Steroidogenesis in Leydig Cells.

The initial steps of steroidogenesis occur in the mitochondria. Dynamic changes in the mitochondria are associated with their fission and fusion. Therefore, understanding the cellular and molecular relationships between steroidogenesis and mitochondrial dynamics is important. The hypothesis of the current study is that mitochondrial fission and fusion are closely associated with steroid hormone synthesis in testicular Leydig cells. Steroid hormone production, induced by dibutyryl cAMP (dbcAMP) in Leydig cells, was accompanied by increased mitochondrial mass. Mitochondrial elongation increased during the dbcAMP-induced steroid production, whereas mitochondrial fragmentation was reduced. Among the mitochondrial-shaping proteins, the level of dynamin-associated protein 1 (Drp1) was altered in response to dbcAMP stimulation. The increase in Drp1 Ser 637 phosphorylation correlated with steroid hormone production in the MA-10 Leydig cells as well as in the primary adult rat Leydig cells. Drp1 was differentially expressed in the Leydig cells during testicular development. Finally, gonadotropin administration altered the status of Drp1 phosphorylation in the Leydig cells of immature rat testes. Overall, mitochondrial dynamics is directly linked to steroidogenesis, and Drp1 plays an important regulatory role during steroidogenesis. This study shows that Drp1 level is regulated by cAMP and that its phosphorylation via protein kinase A (PKA) activation plays a decisive role in mitochondrial shaping by offering an optimal environment for steroid hormone biosynthesis in Leydig cells. Therefore, it is suggested that PKA-mediated Drp1 Ser 637 phosphorylation is indispensable for steroidogenesis in the Leydig cells, and this phosphorylation results in mitochondrial elongation via the relative attenuation of mitochondrial fission during steroidogenesis.

New ionic targets of 3,3′,5′-triiodothyronine at the plasma membrane of rat Sertoli cells

The functions of Sertoli cells, which structurally and functionally support ongoing spermatogenesis, are effectively modulated by thyroid hormones, amongst other molecules. We investigated the mechanism of action of rT3 on calcium (45Ca2+) uptake in Sertoli cells by means of in vitro acute incubation. In addition, we performed electrophysiological recordings of potassium efflux in order to understand the cell repolarization, coupled to the calcium uptake triggered by rT3. Our results indicate that rT3 induces nongenomic responses, as a rapid activation of whole-cell potassium currents in response to rT3 occurred in <5 min in Sertoli cells. In addition, the rT3 metabolite, T2, also exerted a rapid effect on calcium uptake in immature rat testis and in Sertoli cells. rT3 also modulated calcium uptake, which occurred within seconds via the action of selective ionic channels and the Na+/K+ ATPase pump. The rapid response of rT3 is essentially triggered by calcium uptake and cell repolarization, which appear to mediate the secretory functions of Sertoli cells.

Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats.

Cryopreservation of testis tissue is a promising approach to save fertility in prepubertal boys under going gonadotoxic cancer therapies. The using biopolymers as a basis of cryoprotective medium can be effective for the optimization of cryopreservation protocols of immature testicular tissue. The research purpose was to determine morphological parameters and metabolic activity of seminiferous tubules of immature rat testes under exposure to cryoprotective solution (DMSO) based on collagen or fibrin gels (CG or FG) as one of the first stages of developing the cryopreservation protocol. It was found that 30-min exposure of tissue samples to CG and FG with 0.6M DMSO did not impair the spermatogenic epithelium and metabolic activity of the cells (MTT test and total lactate dehydrogenase activity). The use of FG at the time of exposure of 45min did not lead to significant changes in the metabolic activity in contrast to other groups. The findings could be used to substantiate and develop the effective techniques for cryopreservation of immature seminiferous tubules.

Кріоконсервування звитих канальців сім’яників статевонезрілих щурів із застосуванням низьких швидкостей охолодження

Кріоконсервування звитих канальців сім’яників статевонезрілих щурів із застосуванням низьких швидкостей охолодження

Acute effect of bisphenol A: Signaling pathways on calcium influx in immature rat testes

We investigated the acute effect of low concentrations of BPA on calcium influx and the mechanism of action of BPA in this rapid response in the rat testis. BPA increased calcium influx at 1 pM and 1 nM at 300 s of incubation, in a similar manner to that of estradiol. At 1 pM, BPA stimulated calcium influx independently of classical estrogen receptors, consistent with a G-protein coupled receptor. This effect also involves the modulation of ionic channels, such as K+, TRPV1 and Cl− channels. Furthermore, BPA is able to modulate calcium from intracellular storages by inhibiting SERCA and activating IP3 receptor/Ca2+ channels at the endoplasmic reticulum and activate kinase proteins, such as PKA and PKC. The rapid responses of BPA on calcium influx could, in turn, trigger a cross talk by MEK and p38MAPK activation and also mediate genomic responses.

Hormone induced differential transcriptome analysis of Sertoli cells during postnatal maturation of rat testes

Sertoli cells (Sc) are unique somatic cells of testis that are the target of both FSH and testosterone (T) and regulate spermatogenesis. Although Sc of neonatal rat testes are exposed to high levels of FSH and T, robust differentiation of spermatogonial cells becomes conspicuous only after 11-days of postnatal age. We have demonstrated earlier that a developmental switch in terms of hormonal responsiveness occurs in rat Sc at around 12 days of postnatal age during the rapid transition of spermatogonia A to B. Therefore, such “functional maturation” of Sc, during pubertal development becomes prerequisite for the onset of spermatogenesis. However, a conspicuous difference in robust hormone (both T and FSH) induced gene expression during the different phases of Sc maturation restricts our understanding about molecular events necessary for the spermatogenic onset and maintenance. Here, using microarray technology, we for the first time have compared the differential transcriptional profile of Sc isolated and cultured from immature (5 days old), maturing (12 days old) and mature (60 days old) rat testes. Our data revealed that immature Sc express genes involved in cellular growth, metabolism, chemokines, cell division, MAPK and Wnt pathways, while mature Sc are more specialized expressing genes involved in glucose metabolism, phagocytosis, insulin signaling and cytoskeleton structuring. Taken together, this differential transcriptome data provide an important resource to reveal the molecular network of Sc maturation which is necessary to govern male germ cell differentiation, hence, will improve our current understanding of the etiology of some forms of idiopathic male infertility.

Органометрические и морфометрические изменения семенников неполовозрелых крыс в результате искусственной иммуносупрессии

Органометрические и морфометрические изменения семенников неполовозрелых крыс в результате искусственной иммуносупрессии

1,25(OH)2 vitamin D3 signalling on immature rat Sertoli cells: gamma-glutamyl transpeptidase and glucose metabolism.

1α,25-Dihydroxyvitamin D3 (1,25-D3) is critical for the maintenance of normal male reproduction since reduced fertility is observed in vitamin D-deficient rats. Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme that is localized on Sertoli cells and catalyses the transfer of the gamma-glutamyl residues to an amino acid or peptide acceptor. Sertoli cells are also responsible for providing nutrients, as lactate, to the development of germ cells. The aim of this study was to investigate the effect and the mechanism of action of 1,25-D3 on GGT on Sertoli cell functions from 30-day-old immature rat testis. Results demonstrated that 1,25-D3 stimulates GGT activity at Sertoli cells plasma membrane through a PKA-dependent mechanism of action, which was not dependent of active de novo protein synthesis. The hormone increases glucose uptake, as well as lactate production and release by Sertoli cells without altering the reactive oxygen species (ROS) generation. In addition, 1,25-D3 did not change reduced glutathione (GSH) amount or oxygen consumption, and diminished Sertoli cell death. These findings demonstrate that 1,25-D3 stimulatory effect on GGT activity, glucose uptake, LDH activity and lactate production seem to be an important contribution of Sertoli cells for germ cells nutrition and for a full and active ongoing spermatogenesis.

Replacement of serum with ocular fluid for cryopreservation of immature testes

Cryopreservation of immature testis is a feasible approach for germplasm preservation of male animals. Combinations of dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) are used for testis cryopreservation. However, an alternative to FBS is needed, because FBS is expensive. Buffalo ocular fluid (BuOF), a slaughter house by-product, could be an economical option. The objective of the present study was to assess whether BuOF can replace FBS for cryopreservation of immature mouse (Mus musculus), rat (Rattus norvegicus), and buffalo (Bubalus bubalis) testes. Results showed that rodent and buffalo testes frozen in DMSO (10% for rodents and 20% for buffalo) with 20% FBS or BuOF had similar numbers of viable and DNA-damaged cells (P > 0.05). The expression of cell proliferation- (PCNA) and apoptosis-specific proteins (Annexin V and BAX/BCL2 ratio) were also comparable in mouse and buffalo testes frozen in DMSO with FBS or BuOF (P > 0.05). Interestingly, rat testis frozen in DMSO with BuOF had lower expression of Annexin V protein than testis frozen in DMSO with FBS (P < 0.05). The percentage of meiotic germ cells (pachytene-stage spermatocytes) in xenografts from testis frozen either in DMSO with BuOF or FBS did not significantly differ in rats or buffalo (P > 0.05). These findings provide evidence that BuOF has potential to replace FBS for cryopreservation of immature rodent and buffalo testis. Further investigation is needed to explore whether BuOF can replace FBS for testis cryopreservation of other species.

Dose- and time-related effects of caffeine on the testis in immature male rats.

We previously showed that prepubertal chronic caffeine exposure adversely affected the development of the testes in male rats. Here we investigated dose- and time-related effects of caffeine consumption on the testis throughout sexual maturation in prepubertal rats. A total of 80 male SD rats were randomly divided into four groups: controls and rats fed 20, 60, or 120 mg caffeine/kg/day, respectively, via gavage for 10, 20, 30, or 40 days. Preputial separation was monitored daily before the rats were sacrificed. Terminal blood samples were collected for hormone assay, and testes were grossly evaluated and weighed. One testis was processed for histological analysis, and the other was collected to isolate Leydig cells. Caffeine exposure significantly increased the relative weight of the testis in a dose-related manner after 30 days of exposure, whereas the absolute testis weight tended to decrease at the 120 mg dose of caffeine. The mean diameter of the seminiferous tubules and height of the germinal epithelium significantly decreased in the caffeine-fed groups after 40 days of caffeine exposure, which was accompanied by a reduced BrdU incorporation rate in germ cells. In addition, caffeine intake significantly reduced in vivo and ex vivo testosterone production in a dose-related manner. Our results demonstrate that caffeine exposure during sexual maturation alter the testicular microarchitecture and also slow germ cell proliferation even at the 20 mg dose level. Furthermore, caffeine may act directly on Leydig cells and interfere with testosterone production in a dose-related manner, consequently delaying onset of sexual maturation.