Evidence for a role for creatine in male reproductive function

Research into creatine has focussed predominantly on its role in skeletal muscle. However, realisation of the synthetic capacity of many tissues and better understanding of the functions of the CK system has led, in recent years, to considerable interest in the role of the creatine kinase system in non-muscle cells (Wallimann & Hemmer, 1994). In the male reproductive system, this interest has centered on spermatozoa which, like muscle cells, are characterized by intermittently high and fluctuating energy requirements.

Spermatozoa have a DNA-containing head and a long flagellar tail containing the axoneme, which consists of microtubules and dynein. The head and tail are joined by a short midpiece containing mitochondria. The flagellar wave that results in sperm movement is generated by the dynein motor protein which uses ATP as a direct energy source for the movement of microtubules relative to each other. The diffusion of the rather bulky and negatively-charged ATP molecule from the mitochondria to the distal axoneme is likely to be severely limited. However, Tombes and Shapiro (1985) demonstrated that sea urchin sperm, which contain CK levels similar to those found in vertebrate muscle, have distinct mitochondrial and axonemal isoenzymes of CK, leading to the proposal that a phosphocreatine shuttle mediates energy transport from the mitochondrion to the axoneme (Figure 1.8).

Two different CK isoenzymes, brain-type CK (B-CK) and mitochondrial-type CK (Mi- CK), have been identified in rooster and human sperm (Wallimann et al, 1986). In rooster sperm, B-CK was found along the entire length of the tail, whereas Mi-CK was located in the midpiece (Wallimann et al, 1986). Further work has identified the mitochondrial CK isoenzyme in rooster sperm as sarcomeric muscle-type Miy-CK (Wallimann & Hemmer,

1994). However, in human sperm, the identity of the CK located in the mid-piece is not yet clarified, being identified as Mi-CK by Wallimann et al (1986), but as the cytosolic muscle- type M-isoform of CK by Huszar et al (1992). The number of testis-specific isoenzymes already described (Goldberg, 1985), suggests that, indue course, an additional sperm-specific CK isoform may be discovered. The importance of the CK system in spermatozoa is

pH, 7 .0 /h” d y n e i n F L A G E L L U M M O T IL IT Y No pH, 7.5 DYNEIN P , f ADP ATP PC, PCr M ITO C H O N D R IO N TRANSUOCASI ATP ADP ATP FATTY ACIDS R E S P IR A T IO N

Figure 1.8 Schematic of proposed phosphocreatine shuttle in sea urchin sperm. (CK(Mi) = mitochondrial CK; CK(j) = tail CK. Taken from Tombes & Shapiro, 1985.)

demonstrated by the inhibition of rooster sperm motility by CK blockers (Wallimann et al, 1986). In sea urchin sperm, fluorodinitrobenzene (FDNB), which specifically inhibits CK, inhibited coupled, but not uncoupled, respiration and disrupted forward sperm movement by inhibiting flagellar bending in the distal two-thirds of the sperm tail (Tombes & Shapiro,

1985).

The role of the CK system in spermatozoa has, to some extent, been elucidated. However, its presence and role in other cells of the reproductive tract is unknown, as is the source of the creatine and phosphocreatine and the regulatory mechanisms involved. The rat testis has been shown to have the capacity for de novo synthesis of creatine (Alekseeva & Arkhangel’skaya, 1964), which has been shown to be localized within the seminiferous tubular compartment (Moore et al, 1989). Although the cellular distribution of creatine in the male reproductive tract has yet to be fully determined, GAMT in mouse testes has been found to be mainly localized in Sertoli cells of the seminiferous epithelium and microvilli of the initial part of the caput epididymis, but was not detectable in germ cell and interstitial cell-enriched preparations (Lee et al, 1994). Proton NMR analysis of the male reproductive

system in the rat showed high concentrations of creatine in the testes, vasa deferentia, and seminal vesicles, but only lower levels in the caput and cauda epididymes (Navon et al, 1985), suggesting that there may be species differences in the distribution of enzymes involved in creatine synthesis, or that GAMT may have functions other than synthesizing creatine (Lee et al, 1994).

It has been suggested that ecto-protein kinases, found in rat, goat and human sperm, are involved in regulation of sperm forward motility. For this, they would need a high- energy phosphate source (i.e. phosphocreatine) in extracellular semen in order to promote phosphorylation of ADP to ATP (Haider & Majumder, 1986). In mice and rats, Lee et al (1988) have reported the presence of high concentrations of creatine and phosphocreatine in the seminal vesicles. Both creatine and phosphocreatine are secreted into the seminal vesicular fluid, where concentrations are comparable to those in skeletal muscle. This accumulation is regulated by testosterone (Lee et al, 1991). As GAMT mRNA and protein have not been detected in mouse seminal vesicles, it has to be assumed that the creatine is probably transported from the blood into the seminal vesicle lumen (Lee et al, 1994). Human seminal fluid has also been reported to contain high concentrations of creatine (1.3 mmol/L; Geigy Scientific Tables, 1981). Other indications that the external supply of high energy phosphates to vertebrate sperm via seminal vesicle fluid may be important for sperm motility include the discovery that the loss of rooster sperm motility after treatment with CK inhibitors was significantly reversed by the addition of phosphocreatine (Wallimann et al, 1986). In addition, human sperm motility and velocity was enhanced after addition of phosphocreatine in vitro (Fakih et al, 1986). Despite this evidence, it is still unclear whether in vivo phosphocreatine in the seminal fluid can be taken up by spermatozoa and utilized as an immediate energy source, or whether it is metabolized by B-CK, found in both seminal vesicle and prostate fluid, and exerts its effects on sperm motility indirectly (Wallimann & Hemmer, 1994).

Alternatively, creatine might be the product of a pathway used to produce other important compounds, such as S-adenosylhomocysteine, produced in equimolar amounts with creatine due to the action of GAMT, The action of S-adenosylhomocysteine hydrolase, regulated by testosterone in the testis, would then create adenosine able to act locally on Sertoli cell or germ cell receptors. Both adenosylhomocysteine and adenosine have been shown to inhibit sperm motility, and may be used to maintain developing spermatids in a quiescent state within the seminiferous tubule and caput epididymis (Lee et al, 1994).