A commercial human protamine-2 antibody used in several studies to detect mouse protamine-2 recognizes mouse transition protein-2 but not protamine-2 
Matthias Eckhardt and Lihua Wang-Eckhardt

Mol. Hum. Reprod. (2015) 21 (11): 825-831. doi: 10.1093/molehr/gav046

This month’s highlight is a very different subject to usual. Increasingly, we are becoming acutely aware that the repeatability of experiments is not an absolute given. There are a plethora of studies now showing that original findings are not easily repeated for various reasons. One reason is the methods used. How reliable are they? For a number of methods, the cornerstone aspect is a well characterised antibody. 

Eckhardt and colleagues have examined the specificity of a commercially available antibody to human protamine 2. The results are surprising and I won’t spoil the surprise by telling them here. What is clear, though, is that authors and journals need to pay more attention to the methods used for experiments: how robust are they?
In the ESHRE journals, we are meeting these challenges head on by asking reviewers to pay particular attention to the methods section. Importantly we are also open to publication of negative and/or contradictory data, so that the real answers to study questions are uncovered.

Novel characterization of the HSPA2-stabilizing protein BAG6 in human spermatozoa
Elizabeth Bromfield, R. John Aitken, and Brett Nixon
Mol. Hum. Reprod. (2015) 21 (10): 755-769 doi:10.1093/molehr/gav041

Whilst sperm dysfunction is the single most common cause of male infertility currently there is no drug a man can take or that can be added to his sperm in vitro to improve fertility. A fundamental problem with developing new therapies has been the limited understanding of the physiological workings of the normal and dysfunctional spermatozoon. 

In their study, Bromfield and colleagues further examine the molecular details of the complexes on the spermatozoa involved in fertilisation, particularly the interaction with the zona pellucida. Previous studies have identified the key role of a molecular chaperone, HSPA2, and the current paper identifies a role for a further protein, BAG6, which changes localisation during capacitation and has defective expression in the spermatozoa of men with specific sperm-zona abnormalities. The exact function of BAG6 has yet to be determined but a role in the successful expression of HSPA2 is suggested. This work is important as it provides essential detailed molecular characterisation of the sperm interaction with the egg: a black box of knowledge despite 35 years of IVF. 

Functional disparity between human PAWP and PLCζ in the generation of Ca2+oscillations for oocyte activation

Michail Nomikos, Jessica R. Sanders, Junaid Kashir, Randa Sanusi, Luke Buntwal, Daniel Love, Peter Ashley, David Sanders, Paul Knaggs, Adnan Bunkheila, Karl Swann, and F. Anthony Lai
in Mol. Hum. Reprod. (2015) 21 (9): 702-710 doi:10.1093/molehr/gav034 

What in the sperm stimulates the egg to get excited and resume development? A key experiment in this arena is the injection (or production) of sperm factor(s) and subsequent examination of calcium oscillations in the egg. So what is the sperm factor? Until recently the answer was a resounding PLCζ. However another candidate PAWP (a post-acrosomal sheath WW domain-binding protein ) has been proposed as an alternative. The debate is a very topical and sometimes heated. In this paper, Lai and colleagues perform a series of experiments producing, for example, recombinant proteins from both candidates. Significant disparities between previous results using PAWP were observed leading the authors to conclude that PAWP was neither sufficient nor necessary for stimulation of calcium oscillations. The debate is likely to continue and it will be interesting to see results of the knock-out mouse.

Expression and characterization of three Aurora kinase C splice variants found in human oocytes

Jessica E. Fellmeth, Derek Gordon, Christian E. Robins, Richard T. Scott, Jr, Nathan R. Treff, and Karen Schindler 

in Mol. Hum. Reprod. (2015) 21 (8): 633-644 first published online May 20, 2015 doi:10.1093/molehr/gav026

Aneuploidy in oocytes is a fundamental problem in human reproduction. It’s critical we understand the process and consequently what goes wrong. Using information from the mouse as a starting point, Schindler and colleagues, performed a comprehensive study in human oocytes examining the expression and characterisation of Aurora kinase splice variants. They examined these in human oocytes and importantly, using in-vitro experiments, examined the relative importance of each variant. Interestingly, the expression of all three leads to fewer abnormalities in the fidelity of meiosis.

Proteolytic processing of anti-Müllerian hormone differs between human fetal testes and adult ovaries

L.S. Mamsen, T.S. Petersen, J.V. Jeppesen, K. Møllgård, M.L. Grøndahl, A. Larsen, E. Ernst, C. Oxvig, A. Kumar, B. Kalra, and C.Y. Andersen
in Mol. Hum. Reprod. (2015) 21 (7): 571-582. doi: 10.1093/molehr/gav024

A plethora of papers examine the role and potential clinical usefulness of Anti-Müllerian hormone (AMH) in the female. In fact, assessment of AMH has become a centre of attention particularly in ART clinics. The study by Andersen and colleagues takes a different approach; by using a series of antibodies, they examine the distribution of mature and immature forms of the hormone. It turns out that there is a very tight regulation of the processing of AMH which may be key to its different roles in males and females. 

Oxidative stress and human spermatozoa: diagnostic and functional significance of aldehydes generated as a result of lipid peroxidation

Ryan Moazamian, Ashley Polhemus, Haley Connaughton, Barbara Fraser, Sara Whiting, Parviz Gharagozloo, and Robert John Aitken
in Mol. Hum. Reprod. first published online April 2, 2015doi:10.1093/molehr/gav014

Sperm dysfunction, where sperm is present but not fully functional, is the single most common cause of infertility. Yet remarkably, we have no real diagnosis for this condition let alone an effective non-ART treatment. Future developments lie in understanding the function of the normal and the dysfunctional cell. John Aitken and colleagues have pioneered the investigation of oxidative stress in sperm function for the last 25 years. In this study, they present a molecular analysis of the consequences of oxidative stress and suggest a potential robust biomarker for diagnosing this condition. 

PLCζ or PAWP: revisiting the putative mammalian sperm factor that triggers egg activation and embryogenesis

Junaid Kashir, Michail Nomikos, Karl Swann, and F. Anthony Lai
in Mol. Hum. Reprod. first published online February 26, 2015doi:10.1093/molehr/gav009

How does a sperm, at about 1/500,000th the volume of a human egg, stimulate it to wake up and resume activity? The massive differences in volumes are amazing. I think of it as equivalent to a man walking into a football stadium. With this analogy, what’s even more remarkable is that it’s a very small portion of the man which is required for the wake up call, akin to his credit card. But the presence of a credit card is not enough; the egg is only interested in the size of the credit limit. The egg is activated only by a minuscule protein, but it is one which must be very potent. This protein has usually been identified as a variant of phospholipase C (PLC zeta). It's a remarkable protein and the evidence for it being the primary sperm factor is substantial. However, recently another protein has emerged on the scene and although the evidence is less robust, it's gaining traction. In this issue of MHR, Tony Lai and colleagues discuss the fundamental questions surrounding the discovery of sperm factor(s) and point to the critical experiment which is still necessary before a protein can be legitimately described as the key factor for stimulating the egg to wake up.

Intra-sample heterogeneity of sperm DNA methylation Timothy G. Jenkins, Kenneth I. Aston, Cooper Trost, Jordan Farley, James M. Hotaling and Douglas T. Carrell in  Mol. Hum. Reprod. (2015) 21 (4): 313-319. doi: 10.1093/molehr/gau115

The critical question when performing an experiment is how robust is the data. Remarkably often this is ignored. In a detailed analysis, Doug Carrell and his group have examined a fundamental question in sperm biology. What are the differences in the sperm epigenome between ejaculates from the same man and between different men? Reassuringly there were consistent results between and within men. However, interestingly within different populations (good and poor sperm) in an ejaculate, there were clear differences. This suggests that poor quality sperm cells have a different imprint to high quality cells. This is perhaps important food for thought for ART populations. 

Kinetics of human sperm acrosomal exocytosis C.M. Sosa, M.A. Pavarotti, M.N. Zanetti, F.C.M. Zoppino, G.A. De Blas, and L.S. Mayorga in Mol. Hum. Reprod. (2015) doi:10.1093/molehr/gau110

 

The acrosome reaction (AR) is a unique and essential event for fertilisation. However recently there has been a significant reevaluation of where and how the process occurs. We used to believe for example that the AR was induced by the zona. Now we know that the functional AR occurs prior to zona interaction. In this issue of MHR, Sosa and colleagues address the question of how the process occurs. In a beautifully detailed study using a series of techniques such as transmission electron microscopy, the kinetics of this process are revealed for the first time.

1- Subcellular localization of phospholipase C delta in human sperm and its absence in DPY19L2-deficient sperm are consistent with its role in oocyte activation Escoffier et al in Mol. Hum. Reprod. (2014) doi: 10.1093/molehr/gau098

2- Dpyl12-deficient globozoospermic sperm display altered genome packaging and DNA damage that compromises the initiation of embryo development Yassine et al in Mol. Hum. Reprod. (2014) doi: 10.1093/molehr/gau099

This month’s highlight is very different. I’ve picked 2 papers from the same group. Why? These studies address the rare but fascinating pathology known as globozoospermia. Before the discovery of DPLY2 as a deletion that causes globozoospermia (in a significant number of cases) our knowledge of this condition was spare. However now there has been a minor explosion in our understanding of this condition and these two papers make a very significant contribution to the field. They use both a mouse model and humans to try and understand what goes wrong in globozoospermia… fascinating biology. 

The mitochondrion, its genome and their contribution to well-being and disease Justin C. St. John,  Mol. Hum. Reprod. (2015) 21 (1): 1-2. doi: 10.1093/molehr/gau085

This issue of MHR includes a series of articles discussing the increasing importance of mitochondria, not just to reproduction but also to the onset of common diseases. The subject is very topical as there is increasing pressure, due to the severe effects on mitochondrial disorders, to use ART technologies to help relieve the burden of disease. Primarily, this focusses on replacement of the abnormal mitochondria with healthy ones by using, for example, pronuclear transfer. Whilst exciting, these technologies need to be adopted with great care. The reviews in this issue of MHR, including the editorial by Jus St John, highlight the questions that remain to be addressed.