Zhao et al. Reprod Biol Endocrinol (2021) 19:120 https://doi.org/10.1186/s12958-021-00805-1 RESEARCH Open Access Decreased angiotensin receptor 1 expression in AT1 Knockout mice testis results in male infertility± and GnRH reduction Fangfang Zhao1,2†, Yun Zou1†, Hui Li1, Yaheng Zhang1, Xuele Liu1, Xuehao Zhao1, Xinyi Wu1, Wenyi Fei1, Ziling Xu1 and Xuejun Yang1* Abstract Background: This study aimed to detect the efect of angiotensin receptor 1 (AT1) knock out (KO) on spermatogen- esis and hypothalamic-pituitary–gonadal (HPG) axis hormone expression. Methods: Normal C57BL/6 male mice were used as control group or treated with angiotensin receptor blocker, in addition heterozygous AT1KO mice were generated. After caged at a ratio of 2 to 1 with females, pregnancy rates of female mice were determined± by detection of vaginal plugs. Deformity rate of spermatozoa was evaluated by eosin staining and morphology evaluation. The AT1 mRNA expression in the testes of male AT1KO mice was detected by quantitative real-time polymerase chain reaction (QRT-PCR). Serum GnRH level was determined± by ELISA. Results: Compared to control, AT1KO mice showed reduced expression of AT1 in testes, pituitary and hypothala- mus. In addition, decreased level± of GnRH, but not follicle stimulating hormone (FSH) or luteinizing hormone (LH), in AT1KO mice was detected. Treatment with angiotensin receptor blocker (ARB) did not have signifcant efects on HPG± hormones. AT1KO mice exhibited male infertility and signifcant abnormality of sperm morphology. ± Conclusion: Reduced AT1 knockout resulted in male infertility, potentially by inducing abnormal spermatogenesis. Both testis and HPG axis signaling may be involved. Keywords: Angiotensin receptor 1, Male infertility, AT1KO mice, Testis, Hypothalamic-pituitary–gonadal (HPG) axis ± Background be detected. Even in idiopathic normozoospermic male Male infertility is a worldwide problem. Previous studies infertility, a dysfunction of spermatozoa maturation in have shown that male causes accounted for 50% of infer- the female reproductive tract is usually the cause [2]. tility, in which about 30% of cases are still thought to be Tese fndings indicate that abnormal spermatogenesis, idiopathic [1, 2]. In most male fertility cases, abnormal which causes abnormality include sperm production, semen parameters, including azoospermia, oligozoo- quantity, morphology and motility is the main cause of spermia, teratozoospermia, asthenozoospermia could male fertility [2]. Spermatogenesis is a complex process that could be regulated at multiple stages. Although many factors such as genetic, epigenetic, protein expres- *Correspondence: [email protected] sion and post-translational modifcation, as well as envi- †Fangfang Zhao and Yun Zou are co-frst authors and contributed equally to this work ronmental stimulation factors (e.g. oxidative stress) 1 Institute of Nephrology, Shuguang Hospital, Shanghai University have been implicated in abnormal spermatogenesis and of Traditional Chinese Medicine, 528 Zhangheng Road, Pudong New male infertility, however in many cases, the mechanisms Area, Shanghai, China Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhao et al. Reprod Biol Endocrinol (2021) 19:120 Page 2 of 8 causing abnormal spermatogenesis and male infertility mRNA in the brainstem and demonstrated a relation- are still evasive. ship between AT1a and AngII expression. AT1 could be Testis is the location where spermatogenesis and secre- detected in the brainstem and hypothalamus [13]. More- tion of semen occur. Spermatogenesis, which include over, In vitro experiments of human and animal sperma- process of spermatogonia stem cells proliferation or dif- tozoa further showed that AngII could stimulate sperm ferentiation into spermatocytes, diferentiation of sper- motility, increase the level of intracellular calcium in the matocytes, meiotic division, diferenciation of round spermatozoa through AT1, and induce the sperm acro- spermatids and release of mature spermatozoa, mainly some reaction [12, 14]. take place in the seminiferous tubules. Straight seminif- Although the expression of AT1 receptor in central erous tubules are the frst segment of sperm excretion, nervous system, hypothalamus and pituitary has been while leydig cells in the testicular interstitium secrete found for a long time [15, 16], whether it is involved in male hormones. Dysfunction in any of these parts and/ the regulation of GnRH-FSH-LH sex hormones, and or process could cause abnormal semen parameters and whether it is involved in the regulation of spermatogen- male infertility. In addition, the central nervous sex hor- esis and male infrmity through this mechanism has not mone secretion of the hypothalamic-pituitary–gonadal been clarifed and need to be further explored. (HPG) axis plays an important role in the regulation of Te aim of this study is to detect the efects of RAS and spermatogenesis [3]. By puberty, hypothalamic gon- AT1 on male spermatogenesis and reproductive ability adotropin releasing hormone (GnRH) stimulates the by AT1 gene knockout and angiotensin receptor blocker secretion of pituitary gonadotropins, mainly follicle (ARB) treatment. stimulating hormone (FSH) and luteinizing hormone (LH), which stimulate the testis to secrete T allowing Materials and methods for the diferentiation of spermatogonial stem cells, and Animals model establishment the spermatogonia continue to proliferate and ultimately develop into spermatozoa. Male C57BL/6 mice and heterozygous ± AT1KO mice Renin-angiotensin system (RAS) is a hormone sys- were provided by Shanghai Shrek Experimental Ani- tem that plays critical role in blood pressure control [4]. mal Co., Ltd (Shanghai). AT1KO heterozygous mice In recent decades, in-depth research on the RAS has (± AT1KO) model was initially established by the revealed its new biological efects [5], and expression National Natural Science Foundation of China to study of those RAS factors have also been detected in many the role of ± AT1KO in a chronic renal failure model on organs and tissues, suggesting the existence of local- cardiac remodeling and to determine the potential cura- ized RAS functions [6]. Recent studies have shown that tive efect of the traditional Chinese medicine Shenx- renin-angiotensin system (RAS) may be involved in the inning recipe (30,873,262, 2009–2011). Experiment regulation of male infertility and spermatogenesis [7]. male ± AT1KO mice were selected from ofspring of a A large number of gene and protein analyses have con- mating between normal mice and female ± AT1KO mice. frmed the presence of RAS components in the male Te animals were raised in the clean feeding room of the reproductive system [8], including the testis, vas defer- Experimental Animal Center of Shanghai University of ens, epididymis, prostate, sperm, and semen. Te level of Traditional Chinese Medicine. Experimental mice were AngII in semen was found to be 3–5 times higher than divided into the following groups (n = 20 per group): that in the plasma [5]. Group A, normal C57BL/6 mice perfused with normal Te main biological efects of the RAS are largely saline; Group B, normal C57BL/6 mice intragastrically dependent on AngII and through the receptor angio- administered with angiotensin receptor blocker (losartan tensin receptor 1 (AT1) [9]. As documented, the two rat potassium) (ARB group); Group C, male ± AT1KO mice AT1 receptor isoforms (rAT1A and rAT1B) are phar- perfused with normal saline. Ten mice per group were macologically indistinguishable from each other and sampled at 2 weeks and 4 weeks, respectively. Tis study from that of the human (hAT1) [10]. AT1 is expressed was approved by the Shanghai Animal Ethics Review in male reproductive organs and central nervous sys- Committee. tem. It has been reported that the mRNA and protein expression of rAT1A and rAT1B could be detected in Pregnancy test the testis, epididymis, and prostate tissues [11]. Immu- Mice were caged at a ratio of 2 to 1 in males and females nohistochemistry showed the AT1 expression in the at week 2 and week 4 after grouping, and the pregnancy tail of mature spermatozoa and in the head and tail of rate of female mice was determined in one week of cage ejaculated mouse spermatozoa [12]. In addition, Lin and closure (when vaginal plugs were detected, animals were co-workers used in situ hybridization to measure AngII considered to be pregnant). Zhao et al. Reprod Biol Endocrinol (2021) 19:120 Page 3 of 8 Sperm count and morphology analysis Table 1 Primer sequence Te epididymis tissue was harvested, added to 1 ml phos- Gene Primer Synthesized sequence phate-bufered saline, and ground. Te tissue fragments were fltered, and the number of spermatozoa in the sus- mAT1 Forward Primer TTC ATT GAG AAC ACC AAT ATC ACT G pension was counted on a blood cell count plate under Reverse Primer GCT GGT AAG AAT GAT TAG GAA AGG a microscope (Nikon E200, Tokyo, Japan). Te suspen- Probe CGA GTC CCG GAA TTC AAC GCTCC sion was placed on slides and fxed with formaldehyde AngII Forward Primer CGG AAC GAC CTC CTG ACT TG for 5 min.