179 EFFECTS OF DEHYDROEPIANDROSTERONE ON SPERM FERTILIZABILITY IN VITRO AND TESTICULAR GENE EXPRESSION

2012 ◽  
Vol 24 (1) ◽  
pp. 179
Author(s):  
O. Suzuki ◽  
M. Koura ◽  
Y. Noguchi ◽  
K. Uchio-Yamada ◽  
J. Matsuda
Keyword(s):  
Gene Expression ◽  
Internal Control ◽  
Strain Difference ◽  
Strain Differences ◽  
Optimal Dose ◽  
Receptor Protein ◽  
Male Mice ◽  
Vitro Fertilization ◽  
Dhea Treatment

Strain differences of in vitro fertilizability still constitute a serious problem in mouse reproduction. To improve the in vitro fertilizability of mouse sperm, we examined the effects of implanting time-release pellets of dehydroepiandrosterone (DHEA), a testosterone precursor, on sperm fertilizability and testicular gene expression. DHEA pellets (5 mg pellet–1, 21-day release form; Innovative Research of America) or placebo pellets were implanted subcutaneously in 9-week-old male mice from 2 strains: C57BL/6CrNSlc (B6) and 129X1/SvJJmsSlc (129X1). After 21 days, in vitro fertilization was conducted using epididymal sperm from these males and oocytes from superovulated 4-week-old Slc:ICR females. The percentages of 2-cell embryo formation in the placebo and DHEA groups were 78.2 ± 14.2% vs 89.3 ± 2.5%, respectively (mean ± standard error of the mean, n = 4 per group) using B6 sperm and 40.9 ± 8.8% vs 41.3 ± 3.1%, respectively (n = 4 per group) using 129X1 sperm, indicating no significant effect of DHEA treatment (P > 0.05 by 2-way ANOVA). However, a strain difference was quite evident (P < 0.05 by 2-way ANOVA). Quantitative Western blotting using testicular protein extracts with glyceraldehyde-3-phosphate dehydrogenase as an internal control showed that the expression of androgen receptor protein (AR) was significantly higher in B6 than in 129X1 males (P < 0.05 by 2-way ANOVA), whereas there was no significant effect of DHEA on the amount of AR in either strain (P > 0.05 by 2-way ANOVA). In B6 testes, 2-dimensional electrophoresis indicated that 1 protein spot was denser in DHEA-treated males than in placebo-treated males. In 129X1 testes, DHEA did not change the density of the corresponding spot. Mass spectrometry of the spot suggested that it was Cu/Zn superoxide dismutase (SOD1). Thus, 21-day-release pellets of 5 mg DHEA had no significant effect on sperm fertilizability in vitro in 2 strains, but the DHEA pellets induced a strain-dependent testicular protein expression, which might be because of the differential AR expression. Although DHEA treatment of male mice has the potential to improve sperm fertilizability in vitro, a more detailed study is needed to determine the optimal dose, age and duration of DHEA treatment. This work was supported by a grant from the Ministry of Health, Labour and Welfare, Japan.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

Regulation mode of evaporative cooling underlying a strategy of the heat-tolerant FOK rat for enduring ambient heat

2003 ◽  
Vol 285 (6) ◽  
pp. R1439-R1445 ◽  
Author(s):  
Fujiya Furuyama ◽  
Masataka Murakami ◽  
Etsuro Tanaka ◽  
Hideki Hida ◽  
Daisuke Miyazawa ◽  
...  
Keyword(s):  
Heat Tolerance ◽  
Strain Difference ◽  
Strain Differences ◽  
Ambient Temperatures ◽  
Saliva Secretion ◽  
Wide Range ◽  
Heat Tolerant ◽  
Hot Environments ◽  
Regulation Mode

Compared with other rat strains, the inbred FOK rat is extremely heat tolerant. This increased heat tolerance is due largely to the animal's enhanced saliva spreading abilities. The aims of the present study were to 1) quantify the heat tolerance capacity of FOK rats and 2) determine the regulatory mode of the enhanced salivary cooling in these animals. Various strains of rats were acutely exposed to heat. In the heat-intolerant strains, saliva spreading was insufficient and the core temperature (Tc) rose rapidly. In contrast, FOK rats maintained an elevated Tc plateau (39.5 ± 0.7°C) for 5-6 h over a wide range of ambient temperatures (Ta) (37.5-42.5°C). In hot environments the FOK rats secreted copious amounts of saliva and spread it over more than the entire ventral body surface. FOK rats had a low Tc threshold for salivation, and the salivation rate increased linearly in proportion to the Tc deviation from the threshold. No strain difference or temperature effect was observed in the saliva secretion rate from in vitro submandibular glands perfused by sufficient doses of ACh. These results suggest that 1) the ability of FOK rats to maintain a moderate steady-state hyperthermia (39.5 ± 0.7°C) over a wide Ta range is enabled by a lowered threshold Tc for salivation and functional negative-feedback control of saliva secretion and 2) strain differences in ability to endure heat stress are mainly attributable to changes in the thermoregulatory control system rather than altered secretory abilities of the salivary glands.

212 INTERSPECIFIC CLONING AND EMBRYO AGGREGATION INFLUENCE THE EXPRESSION OF oct4, nanog, sox2, AND cdx2 IN CHEETAH AND DOMESTIC CAT BLASTOCYSTS

2015 ◽  
Vol 27 (1) ◽  
pp. 196
Author(s):  
L. N. Moro ◽  
D. Veraguas ◽  
L. Rodriguez-Alvarez ◽  
M. I. Hiriart ◽  
C. Buemo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Internal Control ◽  
Gene Expression Analysis ◽  
Early Embryo ◽  
Domestic Cat ◽  
Nuclear Reprogramming ◽  
Acinonyx Jubatus ◽  
Standard Curve

The cheetah (Ch, Acinonyx jubatus) is a species considered globally endangered and cloning is one of the assisted reproductive techniques that can help to preserve it and to study early embryo development. However, the production of cloned felid embryos remains inefficient, probably because of the difficulty to control the process of nuclear reprogramming and obtain adequate gene expression. Embryo aggregation has been demonstrated to improve the cloning efficiency in several species and to normalise cdx2 in the mouse by lowering its expression (Balbach et al. 2010), but it has not been evaluated in felids before. To better understand the effect of interspecific somatic-cell nuclear transfer (iSCNT) and embryo aggregation in nuclear reprogramming, we analysed the expression of oct4, sox2, nanog, and cdx2 in cheetah blastocysts generated by iSCNT, domestic cat blastocysts (Dc) generated by SCNT, and IVF blastocysts as control. To achieve this, domestic cat oocytes were in vitro matured and zona-free SCNT or iSCNT was performed, as previously described (Moro et al. 2014, Reprod. Fertil. Dev.). Zona-free reconstructed embryos were then cultured individually (1X) or two embryo were cultured together (2X) in microwells, in synthetic oviductal fluid (SOF) medium. The experimental groups were Dc1X, Dc2X, Ch1X, Ch2X, and IVF. After 8 days of in vitro culture the blastocysts obtained were stored in RNA-later at –20°C. For gene expression analysis, blastocysts were pooled as follows: Dc1X, 4 replicates of 3 blastocysts each; Dc2X, 4 replicates of 3 blastocysts each; Ch1X, 2 replicates of 2 blastocysts and 1 replicate of 1 blastocyst; Ch2X, 4 replicates of 3 blastocysts each; IVF 3 replicates of 3 blastocysts each. Embryos were treated with a Cells-to-cDNA TM II kit (Life Technologies, Carlsbad, CA, USA) lyses buffer and treated with DNase I (0.04 U μL–1) for genomic DNA digestion. Gene expression analysis was performed by real-time qPCR using the standard curve method. In all qPCRs, GAPDH was used as an internal control. The statistical analysis was performed using a non-parametric Kruskal–Wallis test (P < 0.05). We observed that Dc1X blastocysts overexpressed the 4 genes evaluated respect to the IVF control. However, the gene expression of the aggregated group (Dc2X) was lower for all the genes, achieving the same levels of nanog and sox2 as the IVF blastocysts. The expression of oct4 and cdx2 were also closer to the expression levels of the control in the Dc2X group than in the Dc1X group. With respect to interspecific embryos, the amount of oct4 and cdx2 was also significantly reduced in the Ch2X blastocysts respect to Ch1X blastocysts. Both cheetah groups showed significantly lower expression of oct4, cdx2, and nanog than the IVF control. In conclusion, transcription of pluripotent and early differentiation factors in cheetah embryos was not as efficient as in the domestic cat embryos, probably caused by interspecific transfer. Our study demonstrated for the first time that defects in gene expression of domestic cat embryos can be corrected by embryo aggregation, providing a simple strategy to improve felid cloning.

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