Primates: Factors affecting fertility

Infertility is often a difficult problem to address, not only because of the unwillingness of partners to discover who has the problem, but also because of the multiple causes of infertility, many of which are unknown. As studying infertility in humans is not feasible, the rhesus monkey (Macaca mulatta) works well as a model organism due to similar reproductive patterns[3]. Several genes or proteins that may be relevant in inhibiting fertility or fertilization have recently been identified, both maternally and paternally. These research findings may be useful in the development of new therapies to treat infertility.

Factors affecting male fertilization:

Glycosylation status during epididymal passage and significance in fertility of a 33 kDa glycoprotein (MEF3)

The epididymis is part of the male reproductive system and is divided into five sections: initial segment,proximal caput, distal caput, corpus, and cauda. As the sperm travels through these sections it is constantly modified, particularly through glycosylation, to form the mature sperm. A recent paper by Chandra et al has identified a glycoprotein monkey epididymal fluid protein 3 (MEF3) that becomes present on the sperm as it maturates in the epididymis, suggesting a role in fertility[1].

Epididymis

Figure 1. Location and structure of the epididymis. The sections from the head to tail of the epididymis are : initial segment,proximal caput, distal caput, corpus, and cauda.  [5]

Methods:

Western blots of the epididymal fluid and sperm membranes obtained from the five sections of the epididymis were used to identify glycosylation status of MEF3 through the use of lectins LCA, WGA, and TGP as probes[1].

The acrosomal reaction was induced through the use of centrifuge and shock and the resulting sperm were then incubated with eggs to detect sperm penetration[1].

Results and Significance:

The lectin probes identified the 33 kDa protein on more mature sperm membranes. Through immunoflourescent localization, MEF3 was seen to be localized on the  surface of spermatocytes and spermatids, suggesting it originates in testis. The existence of the 33 kDa glycoprotein on the surface of capacitated and acrosome reacted human spermatozoa in indirect immunofluorescence assay suggests this protein to be significant for fertilization[1].

Factors affecting female fertility

Maternal effect gene depletion blocks early embryogenesis.

Because embryonic genome becomes active after the 2 cell stage, gene products produced from the oocyte support early embryogenesis. These are called maternal effect genes. One example of this is the NLR family, Pyrin domain containing 5 (NLRP5).  Previous studies indicate that the NLRP family plays an important role in female fertility. However, there is very limited data on the effect of NLRP5 on nonhuman primates. Recently, a paper was published on the effect of NLRP5 depletion on embryogenesis in the rhesus monkey.

Methods: They used RNA interference to knockdown NLRP5. This involves the microinjection of long dsRNA which is more efficient than individual siRNA because it can be processed by endogenous Dicer ribonuclease. While long dsRNA can cause global mRNA destruction, this effect is limited is limited in certain oocyte and embryonic cells due to the lack of protein kinase R/IFN pathway.

  • Controlled Ovarian stimulation: Controlled reproductive cycle to obtain large numbers of macque oocytes through follicular aspiration.



Results:  Depletion of NLRP5 leads to developmental arrest at 8-16 cell stage, thus blocking early embryogenesis. The effects were not seen right away because RNAi is a gene knockdown method so differences were only seen when doses of NLRP5 were low enough.

Changes in development in response to dsRNA treatment

Figure 2. Up until Day 4, cleavage divisions were identical in both the controls(row 1 and 2) and the variable group(row 3). However, after then the embryos treated with NLRP5 ds RNA show blocked development.[3]

NLRP5 treatment with dsRNA

NLRP5 treatment with dsRNA

Figure 3. Top Chart: Shows NLRP5 gene knockdown when treated with NLRP5 ds RNA. Bottom chart shows the effect on the control, NL-RP8.[3].

Maternal vascular endothelial growth factor inhibition of pregnancy establishment

Factors inhibiting blastocyst inhibition can also lead to infertility. Maternal endometrial vascular endothelial growth factor (VEGF) is considered important in blastocyst implantation as VEGF-A regulates the integrity and permeability of blood vessels which could play a role in the embryo-endometrium interaction of blastocyte implantation.  A study by Sengupta et al looks at the immunoneutralization of VEGF-A in the rhesus monkey in order to support the hypothesis that VEGF is important in blastocyst implantation in primates by observing their:

1.)  Role in establishment of receptivity

2.)  Role in implantation and trophoblast invasion

For more information on the role of VEGF in blood vessel formation, check out:

VEGF (Vascular Endothelial Growth Factor)

Methods:

Immunoneutralized VEGF-A during day 5 after ovulation (receptive preparation of endometrium) and day 10 after ovulation (implantation window).

Ovulation was checked for by daily serum profiles of estradiol and progesterone (concentrations provided by ELISA) -> Assumed to be 24 hours after a peak rise in serum estradiol in the presence of progesterone.

Female monkeys after mating were injected with either isotype-matched mouse immunoglobulin (IgG = control) or monoclonal mouse antibody against VEGF (anti-VEGF Mab). On day 13 post-ovulation, endometrium tissue was collected.

  • A bioassay, in which iodinated VEGF binds to receptors, was used to determine the neutralizing activity of the antibody.
  • Serum concentrations of free VEGF were detected to see if administering anti-VEGF Mab effected VEGF levels.

Inhibition of pregnancy was determined by detecting levels of mCG(monkey chorionic gonadotrophin).

Results: Injection of anti-VEGF Mab after 5 days and again after 10 days inhibited blastocyst implantation. Changes in VEGF(and its receptors) expression after treatment in early conceptus tissue suggest a role in placental development as well.

Conclusions

These studies have identified MEF3 as a factor affecting fertility in men and NLPR5 and VEGF as factors affecting fertility in women. Previous studies support the results of the Chandra et al paper, by reporting that MEF3 is poorly expressed in patients suffering from infertility[1]. While the evidence is convincing that NLPR5 deletion decreases fertility, the authors are unsure how to utilize this for therapies or its exact function in embryogenesis, though it may be a redundant gene[3]. The role of VEGF in blastocyst implantation was successfully determined[2]. However, more research is required to determine how these new discoveries can be utilized towards therapy.

References:

[1]. Chandra, Abhishek,  et al. “Post-translational modifications in glycosylation status during epididymal passage and significance in fertility of a 33 kDa glycoprotein (MEF3) of rhesus monkey (Macaca mulatta).”Society for Reproduction and Fertility. 135. (2008): 761–770. Web

[2]. Sengupta, J., , et al. “Immunoneutralization of vascular endothelial growth factor inhibits pregnancy establishment in the rhesus monkey (Macaca mulatta).” Reproduction. 133. (2007): 1199–1211.

[3]. Wu, X. “Maternal depletion of NLRP5 blocks early embryogenesis in rhesus macaque monkeys (Macaca mulatta).” Human Reproduction. 24.2 (2009): 415–424.

[4]. http://www.hhmi.org/biointeractive/cancer/vegf.html

[5]. http://www.nature.com/nrg/journal/v10/n7/fig_tab/nrg2529_F1.html

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