The Effects of Evaporative Cooling on Folliculogenesis and Ovarian Hormones during the Estrous Cycle of Dairy Holstein Cows during the Summer Months in Saudi Arabia

Heat stress is identified as a major cause of lower productive and reproductive performance in animal farming. This situation is more common in Saudi Arabia than in other countries because of extremely high ambient temperatures experienced during the summers. The evaporative cooling cow group had significantly increased serum E2 concentrations on days 7 and 14 - 19 of their estrous cycles, and significantly decreased serum P4 concentrations on days 7 - 18 of their estrous cycles. Evaporative cooling plus shade lowered ambient temperatures (41.80 ± 0.74 versus 47.40?C ± 0.84?C) have increased the relative humidity (0.33 ± 0.01 versus 0.24 ± 0.01) and decreased the temperature humidity index (80.24 ± 0.60 versus 84.77 ± 0.68) when compared with shade alone. However, cows under shade alone, compared to cows under evaporative cooling with shade, had a larger number of small (10.04 ± 0.54 versus 4.72 ± 0.58), medium (2.80 ± 0.21 versus 1.79 ± 0.17), and large follicles (6.82 ± 0.28 versus 5.66 ± 0.22). These results demonstrated that while evaporative cooling had positive effects on dairy cows in this experiment, changes could have been more profound, if installations of curtains and fans were implemented, which might need further investigation.

Current Advances in Epigenetic Modification and Alteration during Mammalian Ovarian Folliculogenesis

During the growth and development of mammalian ovarian follicles, the activation and deactivation of mass genes are under the synergistic control of diverse modifiers through genetic and epigenetic events. Many factors regulate gene activity and functions through epigenetic modification without altering the DNA sequence, and the common mechanisms may include but are not limited to： DNA methylation, histone modifications （e.g., acetylation, deacetylation, phosphorylation, methylation, and ubiquitination）, and RNA- associated silencing of gene expression by noncoding RNA. Over the past decade, substantial progress has been achieved in studies involving the epigenetic alterations during mammalian germ cell development. A number of candidate regulatory factors have been identified. This review focuses on the current available information of epigenetic alterations （e.g., DNA methylation, histone modification, noncoding-RNA-mediated regulation） during mammalian folliculogenesis and recounts when and how epigenetic patterns are differentially established, maintained, or altered in this process. Based on different types of epigenetic regulation, our review follows the temporal progression of events during ovarian folliculogenesis and describes the epigenetic changes and their contributions to germ cell-specific functions at each stage （i.e,, primordial folliculogenesis （follicle formation）, follicle maturation, and follicular atresia）.

New Understandings on Folliculogenesis/Oogenesis Regulation in Mouse as Revealed by Conditional Knockout

In comparison to conventional knockout technology and in vitro research methods, conditional gene knockout has remarkable advantages. In the past decade, especially during the past five years, conditional knockout approaches have been used to study the regulation of folliculogenesis, follicle growth, oocyte maturation and other major reproductive events. In this review, we summarize the recent findings about folliculogenesis/oogenesis regulation, including the functions of four signaling cascades or glycoprotein domains that have been extensively studied by conditional gene deletion. Several other still fragmented areas of related work are introduced which are awaiting clarification. We have also discussed the future potential of this technology in clarifying gene functions in reproductive biology.

Antisense Oligodeoxynucleotide Inhibits Expression of Re-com binantPorcine Follicle-Stim ulating Horm one Receptor

To assess the role of follicle stimulating hormone receptor(FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and 125 I FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR CHO cells). An 18 mer phosphorothioate endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18 mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR CHO cells were cultured in 24 well plates (10 5 cells/well) in the absence or presence of 1 20 μmol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for 125 I FSH binding activity. Treatment with 10 μmol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89±0.06 to 1.64±0.08 ng/mg total RNA ( P <0.05). Transfection with lipofectamine and 0.33 μmol/L antisense ODN caused an increase in porcine mRNA from 0.95±0.08 to 1.53±0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 μmol/L) inhibited membrane binding of 125 I FSH by 13.6± 0.8 % ( P <0.05) in 24 h. Treatment of cells with antisense ODN (10 μmol/L) for 48 h resulted in a 76±1.5 % ( P <0.05) inhibition of 125 I FSH binding. In contrast, transfection with lipofectamine and 0.33 μmol/L antisense ODN at 0 h caused a 76.1±1.3 % ( P <0.05) reduction in binding within 24 h. Binding had returned to 52.3±2.3 % ( P < 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane bound FSHR.

Vitamin D3 supplementation influences ovarian histomorphometry and follicular development in prepubertal albino rats

Objective:To evaluate the development of ovarian follicles in female albino rats following vitamin D3 supplementation.Methods:Eighteen prepubertal female albino rats,aged 3-4 weeks,weighing(70.25±9.16)g,were assigned to three groups(n=6 in each group).Group A was treated with 5.00 mL/kg of distilled water and served as the control group,group B was treated with 0.025 mg/kg of vitamin D3 dissolved in distilled water,and group C was treated with 0.125 mg/kg of vitamin D3 dissolved in distilled water.All treatments were administered orally,twice weekly for 28 days.Blood and ovaries were harvested under anaesthesia.Serum vitamin D3 levels were determined by using spectrophotometric method.Ovaries were processed for histology and every10th hematoxylin and eosin stained-section was selected for histomorphometry.The number of follicles at each developmental stage was estimated.Results:Both 0.025 mg/kg and 0.125 mg/kg of vitamin D3 significantly increased serum concentrations of vitamin D3 and calcium(P<0.05),but did not alter inorganic phosphorus concentration(P>0.05).The control group had fewer growing follicles(primary,secondary and antral follicles)and more non-growing follicles(primordial and atretic follicles)when compared with the vitamin D3-supplemented groups(P<0.05).Vitamin D3 at 0.025 mg/kg significantly increased antral follicles and corpora lutea counts(P<0.05).Vitamin D3 at 0.125 mg/kg significantly increased total,primordial and atretic follicles counts(P<0.05),but significantly decreased primary,secondary,antral follicles count,ovarian weight,relative ovarian weight,and ovarian surface area when compared with the control group and rats treated with 0.025 mg/kg of vitamin D3(P<0.05).Conclusions:Vitamin D3 supplementation at 0.025 mg/kg can enhance optimal ovarian follicle recruitment and development in female rats.

Proteome changes of porcine follicular fluid during follicle development

Background:Ovarian follicular fluid influences follicle and oocyte growth,but the fluctuation of its protein content during folliculogenesis has not been comprehensively analyzed.Here we used a shotgun approach and bioinformatics analyses to investigate and compare the proteomes of porcine follicular fluid(pFF)obtained from small(<4 mm),medium(4–6 mm)and large(>6–12 mm)follicles.Results:Follicular fluid samples containing highest estrogen levels were selected as non-atretic from small(SNA:26.1±15 ng/mL),medium(MNA:162±54 ng/mL),and large(LNA:290±37 ng/mL)follicles for proteomic analyses.We detected 1627,1699,and 1756 proteins in SNA,MNA,and LNA samples,respectively.Nearly 60–63%of total proteins were specific to each sample,11–13%were shared in pairwise comparisons,and 247 proteins were shared among all samples.Functional categorization indicated comparable gene ontology(GO)terms distribution per cellular component,molecular function,and biological process categories across samples;however,the ranking of highly significantly enriched GO terms per category revealed differences between samples.The patterns of proteinto-protein interactions varied throughout follicle development,and proteins such as serine protease inhibitor,clade E(SERPINE);plasminogen activator,urokinase(PLAU);and plasminogen activator,urokinase receptor(PLAUR)appeared stage-specific to SNA,MNA,and LNA,respectively.The“complement and coagulation cascades”was the common major pathway.Besides,properdin and fibulin-1 were abundant proteins that appeared absent in LNA samples.Conclusion:This study provides extensive and functional analyses of the pFF proteome changes during folliculogenesis and offers the potential for novel biomarker discovery in pFF for oocyte quality assessment.

Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

In order to assess the impact of mRNA degradation on steady state levels of follicle stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time dependent changes in FSHR mRNA content were determined by nuclease protection solution hybridization assay (NPA) or by qualitative reverse transcription competitive polymerase chain reaction (RT PCR) in cultured hFSHR YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of uridine into total RNA, by 90 % within 1 h in hFSHR Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time dependent decrease in FSHR mRNA content in hFSHR Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

Is bigger better: The association between follicle size and livebirth rate following IVF?

Purpose: To analyze the correlation between lead follicle size at hCG trigger and IVF outcome. Methods: A review of all patients undergoing their first IVF cycle between 1/2005-12/2009 was performed. Four groups were evaluated (<18 mm, 18 - 18.9mm, 19 - 19.9 mm and ≥20 mm), based on the mean diameter of the two largest follicles on day of ovulation trigger (OT);cycle parameters and outcomes were analyzed. Results: 1577 cycles were reviewed. There were no significant differences noted for cycle parameters or outcomes for the 4 groups. However, although LBR was not significantly different, there was a decline noted as lead follicle size increased. Conclusions: Delaying the administration of OT to enhance follicular growth does not appear to improve IVF outcome. Larger lead follicles do not yield a higher percentage of mature oocytes, embryos available for transfer or LBR. A misguided zest to achieve a higher quantity of fertilizable oocytes may impair oocyte and embryo quality.

A Comprehensive Transcriptomic Analysis of Infant and Adult Mouse Ovary

Ovary development is a complex process involving numerous genes. A well-developed ovary is essential for females to keep fertility and reproduce offspring. In order to gain a better insight into the molecular mechanisms related to the process of mammalian ovary development, we performed a comparative transcriptomic analysis on ovaries isolated from infant and adult mice by using next-generation sequencing technology （SOLID）. We identified 15,454 and 16,646 trans- criptionally active genes at the infant and adult stage, respectively. Among these genes, we also identified 7021 differentially expressed genes. Our analysis suggests that, in general, the adult ovary has a higher level of transcriptomic activity. However, it appears that genes related to primordial follicle development, such as those encoding Figla and Nobox, are more active in the infant ovary, whereas expression of genes vital for follicle development, such as Gdj~, Bmp4 and Bmpl5, is upreg- ulated in the adult. These data suggest a dynamic shift in gene expression during ovary development and it is apparent that these changes function to facilitate follicle maturation, when additional func- tional gene studies are considered. Furthermore, our investigation has also revealed several impor- tant functional pathways, such as apoptosis, MAPK and steroid biosynthesis, that appear to be much more active in the adult ovary compared to those of the infant. These findings will provide a solid foundation for future studies on ovary development in mice and other mammals and help to expand our understanding of the complex molecular and cellular events that occur during postnatal ovary development.

The antiestrogen-like activity and reproductive toxicity of 2,6-DCBQ on female zebrafish upon sub-chronic exposure

2,6-Dichloro-1,4-benzoquinone(2,6-DCBQ), an emerging water disinfection by-product, is widely detected in water resources. However, its potential effects on the reproductive system are largely unknown. Here, we investigated the long-term effects of 2,6-DCBQ on gonadal development by exposing zebrafish from 15 to 180 days postfertilization(dpf). Following exposure to 2,6-DCBQ(20 and 100 μg/L), female-specific effects including delayed puberty onset, retarded ovarian growth and breakdown of the zona radiata were observed, resulting in subfertility in adult females. Adverse effects in folliculogenesis disappeared two months after cessation of 2,6-DCBQ administration. In contrast, no adverse impacts were noted in male testes. The effects on females were associated with significant reduction in 17 β-estradiol(E2) level, suggesting a role for 2,6-DCBQ in anti-estrogenic activity. E2 level change in blood was further supported by dysregulated expression of genes( cyp19a1a, fshb, kiss3, esr2b, vtg1, and vtg3) related to the hypothalamic-pituitary-gonad-liver(HPGL) axis. The present study demonstrates for the first time that 2,6-DCBQ induces reproductive impairments in female zebrafish through disrupting 17 β-estradiol level.

Insights into female germ cell biology： from in vivo development to in vitro derivations

Understanding the mechanisms of human germ cell biology is important for developing infertility treatments. However, little is known about the mechanisms that regulate human gametogenesis due to the difficulties in collecting samples, especially germ cells during fetal development. In contrast to the mitotic arrest of spermatogonia stem cells in the fetal testis, female germ cells proceed into meiosis and began folliculogenesis in fetal ovaries. Regulations of these developmental events, including the initiation of meiosis and the endowment of primordial follicles, remain an enigma. Studying the molecular mechanisms of female germ cell biology in the human ovary has been mostly limited to spatiotemporal characterizations of genes or proteins. Recent efforts in utilizing in vitro differentiation system of stem cells to derive germ cells have allowed researchers to begin studying molecular mechanisms during human germ cell development. Meanwhile, the possibility of isolating female germline stem cells in adult ovaries also excites researchers and generates many debates. This review will mainly focus on presenting and discussing recent in vivo and in vitro studies on female germ cell biology in human. The topics will highlight the progress made in understanding the three main stages of germ cell developments： namely, primordial germ cell formation, meiotic initiation, and folliculogenesis.