Decreased Expression of KIFC1 in Human Testes with Globozoospermic Defects

G C A T T A C G G C A T genes Article Decreased Expression of KIFC1 in Human Testes with Globozoospermic Defects Erlei Zhi 1,2,† , Peng Li 1,2,† , Huixing Chen 1,2 , Peng Xu 3 , Xiaobin Zhu 4 , Zijue Zhu 1,2 , Zuping He 5, * and Zheng Li 1,2, * 1 2 3 4 5 * † Department of Andrology, Center for Men’s Health, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University, 100 Haining Rd, Shanghai 200080, China; (E.Z.); (P.L.); (H.C.); (Z.Z.) Department of ART, Institute of Urology, Urologic Medical Center, Shanghai General Hospital, Shanghai Jiao Tong University, 100 Haining Rd, Shanghai 200080, China Oriental Medical Group Jinghua Hospital of Sheng yang, 156 Nanjing Rd, Shengyang 110000, China; IVF Unit, Department of Obstetrics and Gynecology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University, 197 Ruijin Er Rd, Shanghai 200025, China; State Key Laboratory of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University,160 Pujian Road, Shanghai 200127, China Correspondence: (Z.L.); (Z.H.); Tel.: +8621-36126436 (Z.L.); +8621-63240090 (Z.H.) These authors contribute equally to this work. Academic Editor: Paolo Cinelli Received: 25 July 2016; Accepted: 18 September 2016; Published: 27 September 2016 Abstract: Globozoospermia is a rare (prevalence of <0.1%) but severe male infertility condition. In our previous study, we found that robust KIFC1 immunostaining was detected in the human elongating/elongated spermatids during human acrosomogenesis. However, the relationship between the decreased expression of KIFC1 and human globozoospermia remains largely unknown. Testicular biopsies of 30 globozoospermia and 30 obstructive azoospermia patients who underwent infertility evaluation and treatment were utilized in this study. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blots, immunohistochemistry, an in vivo model, and intratesticular injection of small inhibitory RNA (siRNA) against the Kifc1 gene were employed, and sperm abnormalities were evaluated by hematoxylin and eosin (H&E) staining and immunocytochemistry. We revealed that the testicular level of KIFC1 mRNA in globozoospermia was significantly reduced compared with that in obstructive azoospermia, and the KIFC1 protein was barely detectable in testicular specimens in 30% (9 of 30) of patients with globozoospermia. Furthermore, knockdown of the Kifc1 gene in mice increased the percentage of sperm with globozoospermic defects (26.5%). Decreased KIFC1 expression was mainly observed in the testes of patients with globozoospermia at the spermatid stage, which may be useful for counseling and management of such patients. Keywords: KIFC1; globozoospermic defects; male infertility; kinesin14 family 1. Introduction Globozoospermia is a rare (prevalence of <0.1%) but severe fertility disorder characterized by round-headed spermatozoa with malformed acrosomes or a complete lack of acrosomes [1]. According to the percentage of round-headed and acrosomeless spermatozoa per ejaculate, globozoospermia can be classified as classic/total globozoospermia (100%) or partial globozoospermia (<100%) [2]. Notably, the exact molecular mechanisms underlying this condition remain largely unknown. Genes 2016, 7, 75; doi:10.3390/genes7100075 www.mdpi.com/journal/genes Genes 2016, 7, 75 2 of 11 Mutations in several genes are associated with the globozoospermic phenotype; these genes include Mfsd14a [3], Atg7 [4], Smap2 [5], Spaca1 [6], Dpy19l2 [7], Hsp191 [8], Vps54 [9], Gopc [10], Pick1 [11], Agfg1 [12], and Csnk2a2 [13]. Similarly, causative mutations for globozoospermia have been identified in humans, including those in SPATA16 [14], DPY19L2 [15,16], and PICK1 [10]. KIFC1, a member of the kinesin-14 family, was first identified in the mouse brain and embryos, but its levels are highest in adult testes [17]. KIFC1 is the human homolog of kar3 in yeast, ncd in Drosophila, xctk2 in Xenopuslaevis, and Cho2 in rats. Previous studies have found that KIFC1, as a motor protein, participates in acrosomogenesis in invertebrates and mice. For example, KIFC1 is involved in acrosome formation in Eriocheir sinensis [18] and cell morphological changes in Octopus tankahkeei [19]. KIFC1 also drives acrosome formation and cell morphological changes by interacting with the AFS (Acroframosome) and LCx (Lamellar Complex) during acrosomogenesis in Macrobrachium nipponense [20]. Based on the colocalization of KIFC1 and importin β, KIFC1 has been found to be associated with the acrosome from the initial stages of development in mice [21]. In our previous study, we have found that the expression patterns of the KIFC1 gene are changed during human spermiogenesis and that this gene is highly expressed at the spermatid stage [22]. Therefore, we hypothesized that KIFC1 might play an important role in human acrosomogenesis, and that decreased expression of KIFC1 in human testes would lead to globozoospermic defects. In order to investigate the function of KIFC1 in human acrosomogenesis, we examined specimens obtained from testicular biopsies of patients with globozoospermia and obstructive azoospermia, and compared the expression of KIFC1 in the testes of these patients. We also knocked down the Kifc1 gene in testes of 3-week-old mice to determine the role of KIFC1 in regulating acrosomogenesis. 2. Materials and Methods 2.1. Patients and Samples Patients with globozoospermia and obstructive azoospermia (n = 30 and 30, respectively) were recruited between February 2013 and December 2015, and testicular tissue specimens were obtained by biopsy. Exclusion criteria included abnormal karyotype, Y chromosome microdeletion, hormone treatment at the time of biopsy, exposure to alcohol, drugs, or surgery during the previous 3 months, presence of systemic diseases such as diabetes or hypertension, and a history of vasectomy. Prior to biopsy, demographic information was obtained for each patient. Testis sizes were measured by ultrasound examination, and semen was analyzed. Serum levels of follicle-stimulating hormone (FSH), leuteinizing hormone (LH), testosterone (T), prolactin (PRL), and estradiol (E2) were measured by chemiluminescence assay. 2.2. RNA Extraction and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) RNA was extracted using the RNeasy Micro kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instruction. The precipitated RNA was dissolved in 14 µl of RNase-free water, and the RNA concentration was measured at 260 nm in a spectrophotometer, whereas purity was assessed using the A260 /A280 ratio. Samples were stored at −80 ◦ C until use. Reverse transcription was carried out using a kit (Thermo Scientific, Dalian, China) under the following conditions: 42 ◦ C for 60 min, followed by 70 ◦ C for 5 min. The product was stored at −20 ◦ C for PCR, which was performed under the following conditions: 94 ◦ (...truncated)