Page 1 of 38Reproduction Advance Publication first posted on 16 June 2008 as Manuscript REP-08-0167

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Paternal effect on embryo quality in diabetic mice is related to poor sperm quality and

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associated with decreased GLUT expression

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Sung Tae Kim1, Kelle H. Moley1

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Department of Obstetrics and Gynecology, Washington University in St. Louis, St. Louis,

Missouri 63110

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Short title: Paternal effects on embryo quality in diabetic sperm

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Address correspondence to:

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Dr. Kelle H. Moley Department of Obstetrics and Gynecology, Washington University in St. Louis 660 South Euclid Avenue St. Louis, MO 63110 Telephone: (314) 362-1997 Fax: (314) 747-4150 E-mail: [email protected]

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Abstract

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The objective of this study was to determine if sperm quality, fertilization capacity, and

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subsequent embryo development are altered in diabetic male mice and if differences in

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facilitative glucose transporter (GLUT) expression in testis and sperm exist. Using two type 1

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diabetic mouse models, GLUT expression in testis and sperm was determined by Western

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immunoblotting and immunofluorescence staining. To address sperm quality and fertilization

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capacity, computer assisted sperm analysis (CASA) and in vitro fertilization (IVF) were

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performed. GLUT1, 3 and 5 did not change in expression in the testes or sperm between

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diabetic and non-diabetic mice. GLUT8 and GLUT9b were less expressed in testes of both

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diabetic models vs controls. GLUT9a was not expressed in the Akita testis or sperm as

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compared to strain-matched controls. 3 -HSD expression was significantly decreased in the

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Leydig cells from the diabetic mice. Sperm concentration and motility were significantly lower

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in both the diabetics as compared to the control. These parameters normalized in Akita diabetic

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males treated with insulin. In addition, fertilization rates were significantly lower in the Akita

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group (17.9%) and the streptozotocin (STZ)-injected male group (43.6%) as compared to the

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normal group (88.8%). Interestingly, of the fertilized zygotes, embryo developmental rates to

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the blastocyst stage were lower in both diabetic models (7.1%-Akita and 50.0%-STZ) as

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compared to controls (71.7%).

Male diabetes may cause male subfertility by altering

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steroidogenesis, sperm motility and GLUT expression. This is the first study to link a paternal

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metabolic abnormality to a sperm effect on cell division and subsequent embryonic

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development.

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Key words: GLUT8, GLUT9, embryo, diabetes, testis, sperm

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Introduction

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Diabetes has been associated with reproductive impairments in both men and women.

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Male reproductive alterations have been widely reported in individuals with diabetes. Many

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studies have documented abnormalities in testicular function and spermatogenesis in diabetic

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animals (Murray et al., 1983; Scarano et al., 2006; Seethalakshmi et al., 1987). In men affected

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by insulin-dependent diabetes, sperm have severe structural defects (Baccetti et al., 2002),

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significantly lower motility, and lower ability to penetrate hamster eggs (Shrivastav et al.,

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1989). The administration of high doses of streptozotocin (STZ) to male rodents induces a

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decrease in testicular testosterone production (Sanguinetti et al., 1995; Scarano et al., 2006).

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Glucose is an important energy substrate for most mammalian cells. The entry of

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glucose into the cells is facilitated by a family of glucose transporters (GLUTs) that are

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characterized by the presence of 12 membrane-spanning helices and several conserved

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sequence motifs. Currently, there are thirteen members of the facilitative GLUT family,

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GLUT1-12 and the H+ coupled myo-inositol-transporter (HMIT) (Joost et al., 2002; Wood and

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Trayhurn, 2003). The GLUT family of proteins has been subdivided into three classes: class I

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consists of GLUT1-4; class II consists of GLUT5, 7, 9, 11; and class III consists of GLUT6, 8,

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10, 12 and HMIT (Joost et al., 2002; Joost and Thorens, 2001). GLUTs exhibit a high degree of

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sequence homology; however, they differ in their substrate specificity, kinetic characteristics,

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tissue and subcellular distribution, and their response to extracellular stimuli (Joost et al., 2002;

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Riley et al., 2006).

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GLUT8 is a recently cloned member of the class III facilitative transporters and is

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expressed in the blastocyst stage embryo, heart, skeletal muscle, brain, spleen, prostate,

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intestine, and testis (Carayannopoulos et al., 2000; Doege et al., 2000; Gomez et al., 2006).

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Currently, we have confirmed that GLUT8 is highly expressed in the Leydig cells as well as in

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the innermost cells of the seminiferous tubules in the mouse testis and localizes in the

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acrosome, midpiece, and principal piece of the mouse sperm (Kim and Moley, 2007). Human

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GLUT9 was originally cloned from human kidney cDNA (Phay et al., 2000), and we have

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identified and cloned mouse GLUT9 (Carayannopoulos et al., 2004). Sequence analysis has

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shown that GLUT9 is most similar to GLUT11, and both are categorized to the class II

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subgroup that also includes GLUT5 and GLUT7. A unique feature of mGLUT9 is the existence

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of two predominant splice variants: mGLUT9a and mGLUT9b differing only at the N-terminal

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intracytoplasmic tail (Keembiyehetty et al., 2006). We have shown that GLUT9a and GLUT9b

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are expressed in the mouse testis including the Leydig cells (Kim and Moley, 2007). GLUT9a

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strictly localizes in the midpiece, but GLUT9b localizes in the acrosome, midpiece, and

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principal piece of the mouse sperm (Kim and Moley, 2007). We have shown that surface

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GLUT8 expression is regulated by the insulin/insulin-like growth factor-1 (IGF-1) signaling

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pathways through the IGF-1 receptor in the mouse blastocyst (Carayannopoulos et al., 2000;

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Pinto et al., 2002). In different diabetic models, hepatic GLUT8 has shown altered expression

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patterns (Gorovits et al., 2003). We have also reported that GLUT9 protein expression was

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significantly increased in the kidney and liver from STZ-induced diabetic mice compared with

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nondiabetic mice (Keembiyehetty et al., 2006). Although GLUT8 (Chen et al., 2003; Gomez et

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al., 2006; Ibberson et al., 2002; Kim and Moley, 2007; Schurmann et al., 2002) and GLUT9

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(Kim and Moley, 2007) are expressed in the testis and sperm, it is not known whether their

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expression or localization is altered in the diabetic male reproductive system or not. The

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objective of this study was to determine if sperm quality, fertilization capacity, and subsequent

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embryo development are altered in diabetic male mice and if differences in facilitative glucose

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transporter (GLUT) expression in testis and sperm exist.

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Results

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Expression of GLUT8, GLUT9a, and GLUT9b is altered in diabetic testis and sperm

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In order to detect changes in the expression and localization of GLUT1, GLUT3, GLUT5,

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GLUT8, GLUT9a, and GLUT9b in the diabetic testis, RT-PCR as well as immunofluorescence

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staining and Western blot analysis were performed. No changes in GLUT1, GLUT3 or GLUT5

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expression were detected (data not shown) and these findings were consistent with previous

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reports in diabetic rat (Burant and Davidson, 1994). GLUT8 mainly localized in a punctated

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intracellular compartment of the normal testis (Fig. 1A, panel a). However, the overall

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expression of GLUT8 protein was slightly decreased in the STZ-injected testis (Fig. 1A, panel

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b) and markedly decreased in the Akita testis (see box in Fig. 1A, panel c). GLUT9a protein

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was similarly expressed in the normal and STZ-injected testis (Fig. 1A, panels d-e). However,

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GLUT9a was not expressed in the Akita testis (Fig. 1A, panel f). As shown in Fig. 1B and C,

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we confirmed that GLUT9a protein was not expressed in the Akita testis, although GLUT9a

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mRNA was expressed. GLUT9b was less expressed in both of the diabetic testes as compared

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to normal testes (Fig. 1A, panels g-i and 1C).

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Next, we examined GLUT localization in the diabetic sperm by indirect

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immunofluorescence staining. GLUT8 similarly localized in midpiece and principal piece as

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well as the acrosomal region of the normal and both of the diabetic sperm (Fig. 2A, panels a-c).

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GLUT9a localized in the midpiece of the normal and STZ-injected sperm (Fig. 2A, panels d-e),

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but GLUT9a was not detected in the Akita sperm (Fig. 2A, panel f). As shown in Fig. 2B, we

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Page 8 of 38

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confirmed that GLUT9a protein was not expressed in the Akita sperm, similar to the lack of

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expression in the testis. GLUT9b similarly localized in the acrosomal region, midpiece and

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principal piece of the normal and diabetic sperm (Fig. 2A, panels g-i). GLUT9b expression in

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the acrosome and principal piece was significantly lower in the Akita sperm than in the normal

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and STZ-injected sperm (see the arrows in Fig. 2A, panel i). We confirmed GLUT protein

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expression in the sperm by Western blot analysis (Fig. 2B).

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Insulin protein is unchanged in location but decreased in expression in diabetic sperm

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Insulin in the normal and STZ-injected sperm was similarly expressed and localized in

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the acrosomal region, midpiece and principal piece (Fig. 3A, B), but insulin was significantly

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decreased in the Akita sperm (Fig. 3C). Because STZ injection destroys only the pancreas,

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STZ-injected sperm should still express insulin as much as normal sperm. Since the Akita

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mouse has a single amino acid substitution of the insulin 2 gene, Akita sperm might show faint

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staining with insulin.

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GLUT8, GLUT9a and 3 -HSD expression are decreased in isolated diabetic Leydig cells

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As shown in Fig. 4, GLUT8 protein was slightly lower in both of the diabetic Leydig

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cells than in the normal Leydig cells. GLUT9a was also significantly decreased in STZ-

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injected Leydig cells, and was not detected in the Akita Leydig cells. 3 -HSD, which catalyzes

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the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and

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androstenedione), was significantly decreased in both of the diabetic Leydig cells. This result

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provides supporting evidence as to why diabetic males testosterone levels are lower than

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normal males levels (Scarano et al., 2006).

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Sperm concentration, motility, and fertility were compromised in diabetic male mice

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To examine the quality and fertility of the diabetic sperm, computer-assisted sperm analysis

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(CASA) and in vitro fertilization (IVF) were performed. Cauda epididymal sperm

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concentration was significantly lower in both types of the diabetic male mice than in normal

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mice (Fig 5A, panel a). In the diabetic male mice, the percentage of motile sperm was

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significantly decreased (Fig 5A, panel b), and the percentage of motile sperm with progressive

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motility was also decreased by 50% (Fig 5A, panel c). Path velocity, progressive velocity,

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curvilinear velocity, and lateral amplitude were all significantly reduced in the diabetic mice

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(Data not shown). Beat frequency of both types of the diabetic males was same as that of

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normal males. Straightness and linearity of Akita mice were slightly lower than those of STZ-

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injected and normal mice. The percentage of rapidly moving sperm was lower, and ithe

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percentage of static sperm was higher in both types of the diabetic males than in the normal

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males (Fig. 5A, panels d and e). Next, we performed in vitro fertilization (IVF) with normal

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cumulus-oocyte complex and diabetic sperm.

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As shown in Fig. 5B and C, the normal sperm group exhibited 88.8% of the fertilization

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rate, with 71.7% of fertilized embryos developing to the blastocyst stage. On the other hand,

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sperm isolated from STZ-injected mice showed a markedly decreased fertilization rate (43.6%)

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with 50% of the fertilized embryos developing to the blastocyst stage. Sperm isolated from

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Akita mice also showed a significantly decreased fertilization rate (17.9%) with 7.1% of the

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fertilized embryos developing to the blastocyst stage. In fact, control oocytes fertilized with

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STZ-injected and Akita sperm showed increased fragmentation during cleavage stages and

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poor embryo developmental quality.

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Insulin treatment of Akita mice improves sperm motility and concentration

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Insulin-containing or placebo-containing pellets were placed in non-diabetic and

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diabetic Akita mice. Insulin pellet therapy for 7 days significantly increased the sperm total and

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progressive motility (51% and 103% increase respectively) as well as increasing total sperm

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concentration (51%).

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Discussion

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It is well known that the fertility of germ cells is directly linked with glucose

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metabolism. Glucose is required specifically for sperm fusion to zona-free murine oocytes.

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Studies by Urner and Sakkas have established that glucose must be transported into and

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metabolized by the male gamete in order for fertilization to occur (Urner and Sakkas, 1996).

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Moreover, glucose, not fructose or any other hexose sugars, is required in the medium during

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fusion for successful fertilization as well as to assure maintenance of viability of the embryo

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throughout the preimplantation period (Sakkas et al., 1993).

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disturbed in diabetes and responsible in part for infertility(Glenn et al., 2003) (Sexton and

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Jarow, 1997). However, factors involved in the development of male infertility with insulin-

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dependent diabetes are poorly understood. GLUT8 is highly expressed in the testis

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In addition, spermatogenesis is

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(Carayannopoulos et al., 2000; Doege et al., 2000; Gomez et al., 2006; Kim and Moley, 2007)

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and as such may play a major role in substrate delivery to develop sperm. These observations

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led us to focus on GLUT expression in diabetic male mice. The most commonly used animal

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models of diabetes include rodents, dogs, and primates induced by chemical toxins such as

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STZ or alloxan. However, toxin-induced diabetes in mice has been less successful because of

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strain-dependent resistance to STZ (Rossini et al., 1977). We have used two different kinds of

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type 1 diabetic models, STZ-induced and Akita mice. By Western immunoblotting and

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immunofluorescent microscopy, total amount of GLUT8 protein was decreased in the testis,

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Leydig cells, and sperm of both diabetic models. Furthermore, the plasma membrane

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localization of GLUT8 was slightly decreased in the STZ-injected testis and markedly

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decreased in the Akita testis. Recently, we have shown that GLUT9a and GLUT9b are

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expressed in the mouse testis and differentially localize in the sperm (Kim and Moley, 2007).

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As shown in Figs. 1 and 2, GLUT9a protein was not expressed in the Akita testis and sperm,

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although GLUT9a mRNA was expressed. This absence of GLUT9a protein expression in the

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Akita mice may result from some post-transcriptional events regulated by insulin signaling

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which have been previously described (Freeman and Wolf, 1994; Han et al., 1995). GLUT9b

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expression in the acrosome and principal piece was significantly lower in the Akita sperm than

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in the normal and STZ-injected sperm. The present study indicates that expression of GLUT9

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as well as GLUT8 may be regulated by insulin signaling and/or hyperglycemia and may play

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an important role in sperm maturation, and fertilization. Overall, diabetes might lead to the

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alteration of GLUT expression in the sperm and testis.

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These conclusions are consistent with our prior studies in rodent preimplantation

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embryos as well as other studies in human, rodent and ovine placental tissue showing

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decreased glucose transporter expression in models of maternal diabetes (Chi et al., 2000a; Das

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et al., 1998; Eng et al., 2007a; Eng et al., 2007b; Hahn et al., 1998; Moley et al., 1998b).

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Maternal high glucose concentrations lead to subsequent down-regulation of glucose

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transporter expression and result in decreased intracellular glucose, triggering a cell death

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pathway which we have shown is dependent on BAX and p53 expression (Keim et al., 2001;

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Moley et al., 1998a). Recent work has shown that exposing preimplantation embryos to type 2

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diabetic conditions in vitro, specifically hyperinsulinemia and hyperglycemia, leads to

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apoptosis, decreased glucose uptake and decreased expression of GLUT8 protein in the

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blastocyst stage (Chi et al., 2000b; Eng et al., 2007a). This work now suggests that male

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gametes respond in a similar fashion with a change in expression of glucose transporters in

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response to paternal diabetes.

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According to recent reports, insulin is expressed in human ejaculated sperm (Aquila et

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al., 2005) and epididymal mouse sperm (Kim and Moley, 2007), and it might provide an

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autocrine regulation of glucose metabolism by sperm (Aquila et al., 2005). In this study, we

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confirmed these findings by detecting insulin in control and STZ-injected diabetic mouse as

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shown in Fig. 3A and 3B. But insulin detection in the sperm from Akita diabetic mice is fainter

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than that of the normal and STZ-injected sperm.

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substitution in the insulin 2 gene causing misfolding of the insulin protein (Barber et al., 2005).

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Insulin is detected in secretory granules in Akita mice, however it is profoundly decreased in

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concentration, as we showed here (Wang et al., 1999). Interestingly, treating the Akita mice

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with insulin normalized the sperm concentration, and total and progressive motility of the

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mice, suggesting that insulin signaling and increased GLUT9 expression improves sperm

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quality in diabetic mice.

These mice have a single amino acid

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To determine sperm quality and fertilization capacity of sperm from diabetic vs control

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mice, we performed CASA and IVF with normal cumulus-oocyte complex and cauda-

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epididymal sperm. The epididymis imparts sperm maturation by providing a specific fluid

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environment of secretory products from its epithelium and serves as a site for sperm to acquire

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motility and fertilizing potential (Amann et al., 1993; Cooper, 1998). In addition to sperm

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maturation, the epididymis plays a significant role in the transport, concentration, protection,

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and storage of sperm. Thus, the inevitable role of the epididymis in sperm maturation and the

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adverse effects of diabetes on testicular functions are well established. However, there is not

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enough information on the impact of diabetes on the epididymis. As shown in Fig. 5, this study

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supports, in part, that diabetes could lead to reduced sperm motility and fertility as well as

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compromised epididymal functions for sperm maturation. Sperm isolated from STZ-injected

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and Akita males showed markedly low rates of fertilization and development to the blastocyst

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stage. Although litter size of STZ-injected and Akita male groups through the natural mating

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with normal females was significantly lower than that of normal male group, in vivo

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fertilization rate and embryo development were higher than that of in vitro (Table 1). These

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differences probably reflect the different origin of the sperm. For IVF, we used cauda-

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epididymal sperm, which contain a much higher percentage of non-motile sperm. In natural

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mating, motile and good quality sperm are naturally selected from the vas deferens and female

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reproductive tract during ejaculation and before fertilization.

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Previous studies have also demonstrated paternal effects on cell division in human

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preimplantation embryos obtained by assisted reproductive technologies (Menezo, 2006). It

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was first proposed by Renard and Babinet (Renard and Babinet, 1986) that some factor in the

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male gamete acted at the PN stage to affect later steps in embryonic development. They

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demonstrated that proper nucleoplasmic interaction was necessary to allow normal embryo

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development. The exact factor, however, was not clear. In the early 1990s this paternal effect

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idea was brought into the clinical realm when Janny and Menezo reported that the quality of

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sperm did determine developmental potential of IVF embryos, contrary to popular view(Janny

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and Menezo, 1994). It has been speculated that embryo quality may be negatively affected by

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deficiencies in the sperm nuclear genome or sperm-derived cytoplasmic factors, specifically

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the centrosome as well as oocyte activating factor, responsible for calcium homeostasis in the

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oocyte (Tesarik, 2005). Early paternal effects can be detected as early as the 1-cell zygote and

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are associated with poor zygote and embryo morphology and low cleavage speed. This

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pathology is not thought to associated with increased sperm DNA damage, but more likely this

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is due to as of yet unidentified cytoplasmic factors. Centrioles and oocyte activation factors

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have been raised, but it is still speculative. In contrast, one study disputes this hypothesis and

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reports lower blastocyst formation rates in human ICSI procedures with DNA damaged sperm

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(Nasr-Esfahani et al., 2005). This study showed that DNA fragmentation as detected by the

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Comet assay does not preclude fertilization; however, the embryos derived from these sperm

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have a lower potential to reach the blastocyst stage. All these studies are done in ICSI male

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factor patients whose sperm parameters fall below normal World Health Organization (WHO)

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values.

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In this study, the diabetic mouse sperm parameters were also abnormal. Although the

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exact etiology of these abnormalities is not known, the growing evidence indicates that

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oxidative stress is increased in diabetes, due to the overproduction of reactive oxygen species

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(ROS) and decreased efficiency of antioxidant defenses (Giron et al., 1999; Wiernsperger,

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2003). Oxidation of lipids, proteins and other macromolecules such as DNA occurs during the

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development of diabetes (Ohkawa et al., 1979), and mitochondrial DNA mutations have also

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been reported in diabetic tissues, suggesting oxidative stress-related mitochondrial damage

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(Lee et al., 1997). Mammalian sperm cells present a specific lipid composition with a high

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content of polyunsaturated fatty acids (plasmalogens and sphingomyelins). The lipids in

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spermatozoa are the main substrates for peroxidation, and excess amounts of ROS and free

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radicals have adverse effects on sperm motility and fertility (Aitken et al., 1989). Furthermore,

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oxidative damage to lipids and DNA of spermatozoa is associated with declining motility and

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diminished fertility of human sperm (Chen et al., 1997; Kao et al., 1998). In this regard,

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decreased in vitro fertilization rate in STZ-injected and Akita males might result from oxidative

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stress from dead sperm, but this stress should be more or less overcome through in vivo

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selection procedures during ejaculation and before fertilization. In fact, oocytes fertilized with

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STZ-injected and Akita sperm showed fragmentation and poor embryo development, which

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may be also affected by oxidative stress from sperm. The molecular mechanisms of reduced

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spermatogenesis, motility, and fertilization capacity in diabetes, however, remain to be

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elucidated.

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In addition insulin treatment of the Akita mice for 7 days changed the sperm motility

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and concentration. It has been reported that IGF-II and insulin as well as IGF-I promote

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spermatogonial differentiation into primary spermatocytes by binding to the IGF-I receptor

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(Nakayama et al., 1999). It has been also shown that both the sperm plasma membrane and the

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acrosome represent cytological targets for insulin (Kim and Moley, 2007; Silvestroni et al.,

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1992). We confirm here that insulin is present in mature sperm from control and STZ-treated

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mice but not Akita males. All reports suggest that insulin signaling may be important for

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spermatogenesis, sperm maturation, and capacitation, and support our results suggesting that

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insulin-deficient diabetic mice show decreased sperm quality and fertilization rate.

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Interestingly, GLUT9b expression was increased in the insulin treated Akita mice and

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coincided with improved sperm parameters.

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In conclusion, diabetes in two different mouse models results in male subfertility by

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altering spermatogenesis, steroidogenesis, and sperm maturation. Insulin signaling might play

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an important role in GLUT8 and GLUT9 expression during spermatogenesis, steroidogenesis,

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and sperm maturation. The distribution of GLUTs suggests that they may play different roles

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not only in internal glucose movement, but also in the traffic of sugars for the maturation and

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motility maintenance of the sperm during the capacitation and fertilization processes. The

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present study should provide a clue for the treatment of male infertility in patients with

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diabetes.

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Materials and Methods Animals Mice were housed according to Institutional Animal Care and Use Committee and National Institutes of Health guidelines. The 8-week-old B6XSJL F1 and Type 1 diabetic Akita mice, which have significant hyperglycemia resulting from a single amino acid substitution in the insulin 2 gene and are on a C57 black background, were purchased from Jackson Laboratories, Bar Harbor, ME. To generate a chemically induced diabetic model, B6XSJL male mice received four to five injections of STZ (Sigma Chemical Co. Ltd. St. Louis, MO) at a dose of 100 mg/kg (dissolved in sodium citrate buffer pH 4.4). Seven days post-injection, a tail blood sample was measured for glucose concentrations via a Hemocue B glucose analyzer (Stockholm, Sweden). Mice with blood glucose levels 300 mg/dl were selected. After one month, testes and sperm were isolated. The STZinduced B6XSJL mice, the Akita mice and the non-injected B6XSJL mice were all age matched. The Akita mouse is on a C57 black background and in order to used the B6XSJL as a control, we compared C57 black aged match males to B6XSJL males and found no differences in sperm or testes GLUT expression (Data not shown). There were also no difference in sperm parameters using CASA (Data not shown). For this reason we used aged matched B6XSJL non-diabetic mice as the control for both diabetic groups.

Total RNA extraction and RT-PCR Total RNA was extracted using the RNeasy mini kit (Qiagen, Santa Clarita, CA) according to the manufacturer’s instruction. Reverse transcription with oligo(dT) priming was performed to generate cDNAs from 1 µg total RNA using Superscript II (Invitrogen,

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San Diego, CA) following the instruction provided by the manufacturer. DNA amplification was carried out with Taq DNA polymerase (Invitrogen, San Diego, CA) using the primers in Table 1. Amplified fragments were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining.

Immunohistochemistry and Western immunoblot analysis of testes Immunohistochemistry of GLUTs in the testes was performed in Bouin’s fixed paraffin-embedded or frozen sections as previously described (Keembiyehetty et al., 2006; Kim and Moley, 2007). In brief, testes were removed, fixed in Bouin’s Solution overnight and embedded in paraffin.

Testicular sections were incubated with anti-

GLUT8, anti-GLUT9a, or anti-GLUT9b antibodies. The sections were then incubated with a secondary antibody, Alexa Fluor 546 goat anti-rabbit immunoglobulin G. To-Pro-3 iodide was used to stain the nuclei. Western blot analysis was performed on testes as described previously (Kim and Moley, 2007).

Immunofluorescence and western immunoblot analysis of sperm Indirect immunofluorescence of sperm was performed as described previously (Kim and Moley, 2007). In brief, paraformaldehyde-fixed sperm were incubated with anti-GLUT8, anti-GLUT9a, or anti-GLUT9b antibodies. Sperm were then incubated with a secondary antibody, Alexa Fluor 546 goat anti-rabbit immunoglobulin G. To-Pro-3 iodide was used to stain the nuclei. Western blot analysis was performed on sperm as described previously (Kim and Moley, 2007).

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In vitro fertilization Preparation of spermatozoa The cauda epididymis from male was removed at 12.0-13.0 hrs post-hCG injection and quickly transferred into a culture dish containing 500 µl of HTF medium (Specialty Media, Phillipsburg, NJ), then transferred into another dish with 500 µl of HTF medium. Some incisions were made in the cauda epididymis, and gentle squeezing using fine forceps allowed the spermatozoa to swim out. Capacitation was allowed to proceed for 1.5 hrs at 37 , in a 5% CO2 incubator. The concentration of the spermatozoa was determined, and if necessary, adjusted to obtain similar sperm concentrations from normal, STZ-injected, and Akita males.

Preparation of oocytes Adult nondiabetic B6 female mice, which are seven to eight weeks old, were induced to superovulate by i.p. injection of 5 IU pregnant mare’s serum gonadotrophin (PMSG) followed by 5 IU hCG 48 hrs later. Animals were sacrificed 14.0-15.0 hrs post-hCG injection. Oviducts were collected in a 35 mm dish containing 2 ml HTF medium. The cumulus-oocyte complexes (COCs) were recovered by gentle dissection of the oviducts. COCs were transferred to a 200 ul droplet of HTF medium covered with mineral oil.

Insemination and fertilization Capacitated spermatozoa in the range 1×106 to 2×106/ml were added to each insemination droplet with the cumulus oocyte complexes (COCs) and placed in an incubator for 4-6 hrs. Oocytes then were washed two or three times in 100 ul KSOM

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[potassium simplex optimized medium] (Specialty Media, Phillipsburg, NJ) under oil and finally transferred to 50 µl droplets of the same medium. The dishes were incubated at 37oC in 5% CO2 in a humidified incubator. Fertilization was assessed by recording the number of pronucleus and 2 cell embryos 9-10 hrs and 24 hrs after fertilization, respectively. Embryos were observed at 200× magnification under the inverted microscope. To assess the level of parthenogenesis, eggs were incubated under the same conditions but without sperm addition. This level was consistently 0%.

CASA (computer-assisted sperm analysis) The functional analysis of male reproduction was determined by measuring sperm motility of mice using the CASA system (Hamilton-Thorne Research, Beverly, MA). The cauda epididymis was diced, and the sperm were allowed to disperse into the medium for 10 min at 37oC. Among motility parameters measured were the following: Percent motility—the percent of motile sperm within the analysis field divided by the sum of the motile plus immotile sperm within the analysis field; Path Velocity (VAP)—the average velocity of the smoothed cell path, expressed in microns per second; Progressive Velocity (VSL)—the average velocity measured in a straight line from the beginning to the end of the track; Curvilinear Velocity (VCL)—the sum of the incremental distances moved in each frame along the sampled path divided by the time taken for the sperm to cover the track; Amplitude of Lateral Head (ALH)-the amplitude of lateral head displacement corresponds to the mean width of the head oscillation as the sperm swims; Beat Cross Frequency (BCF)—the frequency with which the sperm track crosses the sperm path; Straightness (STR)—the departure of the cell path from a straight line; Linearity (LIN)—

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the departure of the cell track from a straight line.

Placement of insulin pellets LinBitTM insulin implants (LinShin Canada Inc, Ontario, Canada) were subcutaneously inserted under the mid dorsal skin of Akita male mice during anesthesia. According to manufacturer’s instructions (http://www.linshincanada.com), we added 2 implants for the first 20 g in body weight and another implant for each additional 5 g. The same number of blanks was placed for use as controls. Two days post-surgery, a tail blood sample was measured for glucose concentrations via a Hemocue B glucose analyzer (Stockholm, Sweden). Blood glucose levels

100 mg/dl were selected. After one week, the testes

were isolated.

Acknowledgements This work was funded by NIH RO1HD040390 (KHM) and a research grant from the American Diabetes Association (KHM).

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Figure Legends

Figure. 1. A, Immunofluorescence of GLUT8 (a-c), GLUT9a (d-f), and GLUT9b (g-i) in the normal (a, d, g, and j), STZ-injected (b, e, h, and k), and Akita (c, f, i, and l) testis. j, k, and l; negative controls. Bouin’s fixed paraffin-embedded testicular sections were incubated with anti-GLUT8, anti-GLUT9a, or anti-GLUT9b antibodies. The sections were then incubated with a secondary antibody, Alexa Fluor 546 goat anti-rabbit immunoglobulin G (red fluorescence). To-Pro-3 iodide was used to stain the nuclei (blue fluorescence). This experiment was performed over three times per group with at least 5 animals in each group for each experiment. Comparative RT-PCR (B) and Western blot analysis (C) of GLUT8, GLUT9a, and GLUT9b in the normal, STZ-injected, and Akita testis. GAPDH (B) and -actin (C) were used as internal control for RT-PCR and Western blot analysis, respectively.

Figure 2. A, Immunofluorescence of GLUT8 (a-c), GLUT9a (d-f), and GLUT9b (g-i) in the normal (a, d, g, and j), STZ-injected (b, e, h, and k), and Akita (c, f, i, and l) sperm. j, k, and l; negative controls. Paraformaldehyde-fixed sperm were incubated with antiGLUT8, anti-GLUT9a, or anti-GLUT9b antibodies. Sperm were then incubated with a secondary antibody, Alexa Fluor 546 goat anti-rabbit immunoglobulin G (red fluorescence). To-Pro-3 iodide was used to stain the nuclei (blue fluorescence). This experiment was performed over three times per group with at least 5 animals in each group for each experiment. Arrows are directed at GLUT9b expression at the acrosome and principal piece in the Akita sperm. B, Western blot analysis of GLUT8, GLUT9a, and

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GLUT9b in the normal, STZ-injected, and Akita sperm. -actin was used as internal control.

Figure 3. Immunofluorescence of insulin in the normal (A), STZ-injected (B), and Akita (C) sperm. D; negative control. Paraformaldehyde-fixed sperm were incubated with antiinsulin antibody. Sperm were then incubated with a secondary antibody, Alexa Fluor 488 goat anti-guinea pig immunoglobulin G (green fluorescence). To-Pro-3 iodide was used to stain the nuclei (blue fluorescence). This experiment was performed over three times per group with at least 5 mice per group for each experiment.

Figure 4. Western blot analysis of GLUT8, GLUT9a, GLUT9b, and 3 -HSD in the Leydig cells from the normal, STZ-injected, and Akita testis. -actin was used as internal control.

Figure 5. Computer assisted sperm analysis (CASA) and in vitro fertilization (IVF). A, Mean sperm concentration (a), motility (b), progressive motility (c), slow (d), and static (d) motility in normal, STZ-injected, and Akita males. Asterisks indicate significant differences from normal group. B, Percentage of fertilized embryos with normal oocytes. C, Percentage of fertilized embryos developed to blastocyst.

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Table

Table 1. Comparison of litter size Male

Female

Normal STZ-injected Akita a

Litter size 9.3

Normal

2.3a 5.8a

Significantly differ from the normal group (p