Spermatozoa motility in reverse gear? An observation of backward-moving human spermatozoa.

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1 Spermatozoa motility in reverse gear? An observation of backward-moving human spermatozoa. Correspondence Disciplines Medical Sciences Keywords Spermatozoa, Motility Humans Type of Observation Standalone Type of Link Case study Submitted Sep 12, 2016 Published Dec 8, x Triple Blind Peer Review The handling editor, the reviewers, and the authors are all blinded during the review process. Full Open Access Supported by the Velux Foundation, the University of Zurich, and the EPFL School of Life Sciences. Ranjani Sivakumar, Prathima Tholeti, Ramesh Raja Dhanusu, Asha Benziger, Pandiyan Natarajan Dept of Reproductive Medicine, Chettinad Health and Research Institute; Department of Clinical Embryology, KMC, Manipal University; Department Of Reproductive Medicine, Chettinad Health and Research Institute; Department of Reproductive Medicine, Chettinad Health and Research Institute Abstract Motility of the mammalian spermatozoa is characterized by the flagellar waveforms that are propagated from base to tip, which helps them to move forward. We have made an observation of backward-moving spermatozoa in the fresh ejaculate of a patient with normozoospermic sperm parameters. These spermatozoa were observed to have a bend either in the mid-piece or in the principal piece region, but the motility was rapid and comparable to that of spermatozoa with normal structure. We put forth two hypotheses to explain this mechanism of backward motility. However, further studies are required to understand the molecular structure and mechanism. Objective We report an observation of reverse-motile human spermatozoa in the neat sample,that is, fresh ejaculate of a patient who had come for infertility treatment to our hospital. Introduction Physiological motility in the mammalian spermatozoa, which is an inherently motile cell, is of two types - activated (fresh ejaculate) and hyperactivated (site of fertilization) - both of which are characterized by flagellar wave forms, symmetric or asymmetric, but definitely forward progressive, that is head first [1]. In a few animal species,however, the spermatozoa have been described to move in a reverse direction (i.e. tail first), like in certain species of snails, fruit flies, and myzomotoid worm [2] [3] [4]. The mechanism of this phenomenon has not been completely understood yet;this in spite of theories of presence of two kinetic centers for oscillation and reversal of rotational direction [2] [3]. In 1983, Phillips and Kalay reported an observation of backward-swimming spermatozoa in a mammalian species. They observed hairpin-shape spermatozoa which progressed backward in cryopreserved- thawed bull semen and concluded that, in mammalian spermatozoa, a wave can be propagated in a base-to-tip manner even by the caudal end of the flagella, not necessarily only from the basal end, resulting in the backward motion (Phillips and Kalay, 1983) [5]. 4.0 Creative Commons 4.0 This observation is distributed under the terms of the Creative Commons Attribution 4.0 International License. DOI: /matters Matters (ISSN: ) 1

2 Spermatozoa motility in reverse gear? An observation of backward-moving human spermatozoa. a b DOI: /matters Matters (ISSN: ) 2

3 Spermatozoa motility in reverse gear? An observation of backward-moving human spermatozoa. c d DOI: /matters Matters (ISSN: ) 3

4 Spermatozoa motility in reverse gear? An observation of backward-moving human spermatozoa. e Figure Legend Fig. (A) : Tail bent in the mid-piece region Fig. (B, D, E) : Tail bent in the principal piece region Fig. (C 1) : Base-to-tip wave propagation in normal mammalian spermatozoa Fig. (C 2) : Hypothesis A - Base-to-tip propagation of wave but due to the bent tail (head at the side of the tail and not at the base) it appears to be moving in a reverse direction Fig. (C 3) : Hypothesis B - Tip-to-base propagation of wave by the distal end of the flagellum due to a structural defect. Note: Blue thin arrows indicate wave propagation; red thick arrows indicate progression of sperm Results & Discussion We report an observation of backward motile human spermatozoa in the neat sample, that is, fresh ejaculate of a patient who had come for infertility treatment to our hospital. The patient was a 35-year-old marketing executive with an infertility duration of 54 months. The sample was collected through masturbation and was normozoopermic according to WHO 2010 criteria [6] (Sperm concentration: 71 million/ml; total motility: 68%; normal morphology: 4% and normal physical examination (testis volume: left cc; right - 11 cc), vasa felt on both sides and both cords were bulky. The patient had a history of smoking and alcohol consumption for the last 10 years. In this patient, backward motile spermatozoa were observed in two consecutive ejaculates obtained within a gap of 2 months. The spermatozoa were observed in the wet preparation at 40x highpower field (HPF); of the 68% motile spermatozoa, 10% showed reverse motility. All of the backward motile spermatozoa were seen to have structural defects in the tail region, where it was bent back on itself in a sharp fold, forming a deep- U shaped tail (Fig. A, B, D, E). In some spermatozoa, the fold was seen in the mid-piece region, while in a few others it was in the principal piece region. The motility, however, was not compromised in spite of such a structural defect (Normal: 4%; Head Defects: 59%, Mid-piece defect: 2%; Tail defect: 0%; multiple defect: 35%). On the contrary, it was comparable to that of structurally normal motile spermatozoa, only it appeared to be moving backward (S1: Video 1). The couple came with secondary infertility; the first pregnancy was a spontaneous conception, but had a miscarriage in 50 days of conception. Hypotheses: The structure of the flagellum, including the finer details, has been elucidated in detail, but the mechanism of spermatozoa motility is still not completely understood. The identification of dynein, the motor protein [7], on the microtubule doublets of the axoneme led to further studies attempting to understand the molecular mechanisms of motility [8] [9]. It has been accepted that the dynein proteins causes sliding of outer microtubule doublets, but due to the resistance provided by the nexin links, the sliding is converted into a bend formation in the tail, which when repetitive propagates a wave from base to tip. This unidirectional wave propagation in most species is because of the alternate activation and inactivation cycles along the length of the axoneme of the dynein motor protein - a minus (-) end- directed protein that pushes the microtubules to the plus end (+) (tip of the flagellum) [1] [10]. In cases of bent tails such as the ones reported in this study, we put forth two hypotheses for the mechanism of reverse motility: 1. The wave propagation is base to tip (Fig. C1), but as it is the tail, and not the head, that is seen leading the cell (due to structural deformity), it appears that the sperm is moving in the reverse direction. In actuality, the spermatozoa could be progressing forward, DOI: /matters Matters (ISSN: ) 4

5 only the head is at the side of the tail and not at the front (Fig. C2). 2. Due to structural defect, the wave may propagate in a tip-to-base manner akin to that seen in kinetoplastid spermatozoa. In these spermatozoa, the axonemal assembly is the same as that found in other organisms whose wave propagation is unidirectional, yet the direction the wave propagates can be changed based on the circumstances [11]. Similarly, in the spermatozoa that we have observed the structural defect may have caused the reversal of wave propagation (tip to base), but the plausibility of this needs an in-depth molecular study of these bent flagella (Fig. C3). However, the explanation put forth by Phillips and Kalay(1983) [5] cannot be ignored. They state that in such bent-tail spermatozoa the flagella at the bent portion are damaged and therefore do not propagate a wave; instead, the distal end takes up that function. This autonomous functional capacity by any given part of the flagellum has been demonstrated by Brokaw(1961) [12] and also by Lindemann and Rikmenspoel(1972) [13], in spermatozoa of other invertebrate organisms. Several studies have been conducted to confirm that this characteristic phenomena may be valid to axonemes of all species [14] [15]. However, whether the backward motility is because of the hairpin shape cannot be ascertained as not all spermatozoa with such structural defects move backward, even within the same sample. At the same time, the absence of reports in the literature of backward swimming human spermatozoa with normal structure makes it more intriguing. Also worth mentioning is the rarity of this phenomenon in other patients with similar structural defects. Forward progressive motility of the spermatozoa is one of the most important parameters that is analyzed apart from sperm concentration and morphology. The observation of backward motile spermatozoa is however intriguing. Further research is needed to understand the ultrastructure and the molecular mechanisms of these spermatozoa in order to better understand this backward movement of spermatozoa, whose inherent nature is to progress forward toward the oocyte. Limitation of the study: 1. Sample size includes only one subject who had backward-moving spermatozoa in consecutive ejaculates. 2. As this observation was made in a routine andrology laboratory of an IVF clinic, there is no access to high-resolution microscopy/ imaging software, hence the poor-resolution pictures taken while performing standard semen analysis. Also further research could not conducted due to the same reason. The observation was made in a routine andrology laboratory and the sample was not processed. Reverse-moving spermatozoa was observed in the neat sample. So further research has to be conducted to know why only 10% of the spermatozoa was backward moving and also about acrosome reaction possibilities. Additional Information Methods and Supplementary Material Please see No funding was sought for this study. The authors acknowledge the Department of Reproductive Medicine, Chettinad Super Specialty hospital. Ethics Statement Not Applicable. Citations [1] Turner and R. Tales from the Tail: What do we really know about sperm motility? In: Journal of andrology 24(6) (2003), pp [2] Ishijima et al. Movement of Turritella spermatozoa: Direction of propagation and chirality of flagellar bends. In: Cell Motil. Cytoskeleton. 44(2) (1999), pp

6 [3] Buckland-Nicks et al. Locomotion of the filiform sperm of Littorina Gastropoda, Prosobranchia. In: Cell and Tissue Research (1981), pp [4] Ishijima et al. Movement of Myzostomum spermatozoa: Calcium ion regulation of swimming direction. In: Cell Motil. Cytoskeleton. 28(2) (1994), pp [5] Phillips and D.M. Mechanisms of flagellar motility deduced from backward swimming bull sperm. In: J Exp Zool 231(1) (1984), pp [6] World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen. In: 5th ed. Geneva: WHO Press (2010). [7] Gibbons et al. Dynein: A Protein with Adenosine Triphosphatase Activity from Cilia. In: Journal of Science 149 (1965), pp [8] Lindemann and C.B. The geometric clutch as a working hypothesis for future research on cilia and flagella. In: Ann. N. Y. Acad. Sci 1101 (2007), pp [9] Woolley and D. M. Flagellar oscillation: a commentary on proposed mechanisms. In: Biol. Rev. Camb. Philos. Soc. 85 (2010), pp [10] Inaba and K. Sperm flagella: comparative and phylogenetic perspectives of protein components. In: Mol. Hum. Reprod. 17 (2011), pp [11] Surgue et al. Flagellar wave reversal in the kinetoplastid flagellate Crithidia oncopelti. In: Biology of the Cell 63(2) (1988), pp [12] Brokaw and C. J. Movement and nucleoside polyphosphatase activity of isolated flagella from Polytoma uvella. In: Exp. Cell Res 22 (1961), pp [13] Lindemann et al. Sperm flagellar motion maintained by ADP. In: Expl Cell Res. 73 (1972), pp [14] Okuno et al. Mechanical stimulation of starfish sperm flagella. In: J. exp. Biol. 65 (1976), pp [15] Hayashibe et al. Induction of temporary beating in paralyzed flagella of Chlamydomonas mutants by application of external force. In: Cell Motil. Cytoskeleton. 37 (1997), pp