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Studying the effect of seasonal heat stress on the ovarian pool of oocytes is an enormous

challenge. In early studies, we characterized the effects of elevated temperature on the oocyte's

capacity to undergo maturation, fertilization and further development to a viable embryo.

Using a time-lapse system, we were the first to show that seasonal heat stress delays the first

two embryonic cleavages along with decreased embryonic development. In-vitro exposure of

oocytes to physiologically relevant thermal stress impaired cytoskeleton rearrangement and spindle-apparatus formation, reduced the proportion of oocytes progressing to metaphase II, and induced apoptosis, via the sphingomyelin pathway, a transmembrane signaling system.

In a pivotal study, we documented that in-vitro heat-shock simulates the effects of heat

stress that animals experience in vivo. The most prominent finding was that in both models,

the relative transcript abundance of the examined genes (MOS, GDF9, GAPDH, POU5F1) was

similarly reduced in heat-stressed oocytes. We provided first evidences that during the summer

POU5F1 mRNA expression is reduced throughout all embryonic developmental stages, before and

after embryonic genome activation. POU5F1 is a member of the POU family of transcriptional

activators which are essential for maintenance of totipotency/pluripotency in embryonic stem cells.

These findings support the concept that heat stress induces disruption of RNA storage in GV-stage

oocytes and renewed transcription in mature oocytes. Further support stems from new evidence,

which we recently uncovered, of seasonal alterations in RNA expression of mitochondrion-associated genes (ND2, SDHD, CYTB, COX3, B5ATP, TFAM) involved in the electron-transport chain through mitochondrial ATP production. These alterations were associated with changes in mitochondrial distribution and membrane polarity. Interestingly, incorporation of coenzyme Q10, a ubiquitous free-radical scavenger and a key component of the mitochondrial respiratory chain, increased the

proportion of polarized mitochondria and improved embryonic development. These findings significantly contribute to our understanding that heat-stress induced mitochondrial dysfunction involves in the mechanism disrupts of oocyte competence. 

Effect of thermal stress on the follicle-enclosed oocyte
Hormonal treatment to improve fertility in heat-stressed dairy cows

Our findings have indicated both immediate and carryover effects on the follicle and its enclosed oocyte. This concept is now well-accepted and based on that, we developed a novel in-vivo approach in which frequent follicular aspiration enhanced removal of summer-impaired follicles. Years later, these findings have served to develop hormonal-treatment strategies for improving fertility in dairy cows during the summer. The most powerful approach is based on induction of short follicular cycles by GnRH and PGF2a to enhance the emergence of a healthy preovulatory follicle before artificial insemination. A second approach is based on inserting a controlled intravaginal drug release device containing progesterone to support the embryo in its early stage of development. As a consequence, we recently reported that combining these two treatments into continuous administration, before and after artificial insemination, had significant beneficial effects on large population of cows. These hormonal approaches can be easily incorporated into reproduction-management practices on commercial dairy farms in Israel and in other hot regions of the world. 

Association between seasonal fertility and semen quality

Decreased fertility of dairy cows during the summer is also associated with impairment of the male gamete. Therefore, studying the quality of semen is one of my new research interests. Early on, we documented reduced mRNA expression for very-low-density lipoprotein receptors during the summer, suggesting modifications in sperm extracellular lipid utilization. In another study, we showed the inferiority of summer semen for cryopreservation. Post-thaw examinations revealed a reduced motility, impaired progressive motility (PM), and a higher proportion of sperm with damaged acrosomes. We found that semen functional traits varied between seasons in association with differential alterations in lipid composition among sperm compartments. Alterations in the sperm tail expressed by high saturated fatty acids (FAs), low polyunsaturated FAs and low cholesterol concentrations, in association with reduced PM.

PM is the sperm’s ability to move straight forward in a clearly defined direction in the female reproductive tract. Comparison between high- and low-PM spermatozoa revealed a correlation between high PM and high in-vitro fertilization capacity. Recently, we provided first evidences for a high proportion of omega-3 and low proportion of omega-6 fatty acids in high-PM semen, suggesting that lipid composition is involved in the mechanism underlying PM. In light of these findings, we proposed that changing the membrane lipid composition by nutritional means will further improve sperm PM. This assumption is strongly supported by our new findings that feeding a diet enriched in omega-3 fatty acids changes the proportion of C22:6n3 (DHA) in the sperm and increases PM. 

Effects of endocrine-disrupting chemicals on oocyte function

Developmental competence of oocytes and preimplantation embryos can be inhibited by a range of chemicals, including ingredients in foodstuffs and environmental contaminants. In the framework HU-CEAEH we examine the risks associated with phthalate exposure to bovine oocyte. Phthalate esters are a class of water-insoluble synthetic organic chemicals widely used in industrial applications, and known as endocrine-disruptive compounds (EDCs).

We established an in-vivo model to examine the clearance pattern of Bis(2-ethylhexyl) phthalate (DEHP) metabolites from the urine, milk and plasma. The model enabled defining two phases:  acute exposure of 50 µM mono-(2-ethylhexyl) phthalate (MEHP), which is considered toxic, and a relatively low level of 20 nM MEHP which is more physiologically relevant. Acute exposure of cumulus oocyte complexes to MEHP have a multifactorial effect on the oocyte, as reflected in both the nuclear and cytoplasmic maturation mechanisms, which eventually led to a reduction in oocyte developmental competence. The disruptions included alterations in meiosis resumption, cortical granules and endoplasmic reticulum reorganization. MEHP also impaired mitochondrial functioning, increased level of intracellular reactive oxygen species and increased proportion of apoptotic oocytes. Using trans-rectal ultrasound monitoring, we have recently documented a disruptive carryover effect of DEHP on ovarian function. In particular, it attenuated growth rate and reduced estradiol concentration in the preovulatory follicular fluid.  Furthermore, we detected 20 nM MEHP in the follicular fluids obtained from DEHP-treated cows. Accordingly we established an ex-vivo model by which oocytes were in-vitro matured in follicular fluids containging residual low concentration of MEHP. We found that culturing in low MEHP concentrations redues oocyte developmental competence. In addition, low MEHP found to alter the expression levels of fundamental developmental, mitochondrial and epigenetic genes in matured oocytes as well as in blastocyts that developed from MEHP-treated oocytes indicating a carry over effect of MEHP. These findings provide first evidence that exposure to minute doses of EDCs has long-term detrimental effects These emerging data in farm animals must also be carefully considered in evaluating potential risks to humans.

Effects of endocrine-disrupting chemicals and foodborne contamination on sperm

The gradual decline in fertility of human and farm animals over the past few decades

coincides with intensive industrial and agricultural development. Multiple environmental factors affect sperm function and fertility. This project focuses on the direct effects of foodborne toxins on spermatozoa while exploring the potential hazards associated with aflatoxin B1 (AFB1) and Atrazine exposure.  Aflatoxins are poisonous byproducts of the soilborne fungus Aspergillus, involved in the decomposition of plant materials. Aflatoxins can be found in various food products, such as maize, sorghum, millet, rice and wheat. AFB1 is the most toxic of these, classified as a carcinogen and mutagen for both humans and animals.  We found that exposure of

sperm to low concentrations of AFB1 for a few hours resulted in decreased

sperm viability and hyperpolarization of the mitochondrial membrane, most

markedly with ejaculated sperm, suggesting that not only the earlier stages

of spermatogenesis are affected.

 

Exposure of fresh semen to AFB1 prefertilization resulted in sperm DNA

damage and impaired fertilization competence, expressed by reduced

proportion of oocytes that cleaved to 2- and 4-cell stage embryos

Atrazine (ATZ), one of the most extensively used herbicides, is considered a

ubiquitous environmental contaminant and it is a known as EDCs that 

deleterious affects reproductive function. Once it enters the body,

ATZ is metabolized to various metabolites, which are further detected in the

urine, serum and tissues. In mammals, the major ATZ metabolite is diaminochlorotriazine (DACT). We examined the direct effects of low doses of ATZ and DACT on bovine sperm isolated from ejaculates or epididymis compartments (head, body and tail). 

The findings indicated that both ATZ and DACT adversely affect sperm, expressed by damaged sperm membranes. ATZ had a prominent effect on epididymal-tail sperm, expressed as disruption of all examined membranes, mostly at low concentrations; pseudo acrosome reaction and that induced by Ca++ ionophore were both affected by exposure ATZ. A similar pattern was documented for sperm isolated from ejaculates. ΔΨm was affected by ATZ in sperm isolated from the epididymis tail, but not in that isolated from ejaculates. DACT reduced sperm viability at all examined concentrations and in all fractions. DACT at 1 μM impaired ΔΨm in sperm isolated from the epididymis tail and ejaculate. Induction of AR by Ca++ ionophore was impaired in sperm isolated from ejaculate and exposed to 10 or 100 μM DACT and in sperm isolated from the epididymis tail and exposed to 1, 10 or 100 μM DACT. These findings reveal the harmful effect of exposure to ATZ and DACT, mainly at low ecologically relevant doses, on sperm viability, AR and mitochondrial function. We conclude that sperm at advanced stages of spermatogenesis, through its passage and storage in the epididymis compartments as well as in the ejaculate, is sensitive to herbicide. The results suggest that ATZ- or DACT-induced disruptions of sperm membranes might impair sperm fertilization competence.

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