围生期低剂量双酚A对子代雄鼠生精功能的影响

doi:10.12280/gjszjk.20210097

摘要/Abstract

摘要：

目的: 研究围生期低剂量双酚A（BPA）对子代雄鼠睾丸生精功能的影响。方法: 妊娠母鼠在妊娠第10天到分娩后第7天,一组每天皮下注射2 μg/kg BPA（其雄性子代鼠为BPA染毒组）,另一组妊娠母鼠每日皮下注射等体积的溶剂橄榄油（其雄性子代鼠为对照组）。子代雄鼠分别在出生后18、21、24 d处死,观察睾丸组织学结构、检测睾丸内cyclin A1、cyclin B、c-jun和c-fos基因的表达,测定血浆卵泡刺激素（FSH）和黄体生成激素（LH）的水平,检测下丘脑视前区（POA）组织促性腺激素释放激素（GnRH）和弓状核（ARC）组织kiss1的mRNA表达。结果: 24日龄BPA组子代雄鼠的睾丸生精小管直径、圆形精子细胞数量以及圆形精子细胞管腔率较对照组增加（P<0.05）。21日龄BPA组子代雄鼠睾丸内cyclin A1、c-jun和c-fos的基因表达高于对照组（均P<0.01）。BPA组子代雄鼠出生后21、24 d的血浆FSH和LH的水平均高于对照组（均P<0.05）。然而2组的血浆睾酮（T）和雌二醇（E₂）水平差异无统计学意义（P>0.05）。同时,21日龄BPA组子代雄鼠的下丘脑POA-GnRH和ARC-kiss1的mRNA表达水平高于对照组（GnRH：P<0.05;kiss1：P<0.01）。结论: 围生期低剂量BPA长时间暴露除直接作用于睾丸影响精子发生外,还通过激活ARC-kisspeptin神经元,提前启动生殖内分泌轴,使睾丸精子发生提前。

关键词: 精子发生, 围产期, 减数分裂, 下丘脑, 动物实验, 大鼠, 双酚A

Abstract:

Objective: To study the influence of perinatal exposure of bisphenol A (BPA) at a low dose on the spermatogenesis of male offspring rats. Methods: Pregnant rats were injected subcutaneously with BPA 2 μg/(kg·d) from gestation day 10 through lactation day 7, and the same volume of olive oil was used as the blank control. Their male offspring were served as the BPA group and the control group. Histological structure of the testis and the related regulation genes such as cyclin A1、cyclin B、c-jun and c-fos, as well as the FSH and LH, were tested, and the expressions POA-GnRH or ARC-kiss1 mRNAs were also examined in their male offspring during the postnatal day 18, 21, 24. Results: In comparison with the control group, the seminiferous tubular diameter, the number of round spermatids and the ratio of seminiferous tubule with round spermatids were significantly increased in PND24 BPA rats (P<0.05). The expression levels of cyclin A1, c-jun and c-fos mRNAs in the testes were significantly elevated in PND21 BPA rats (P<0.01). Similarly, the levels of plasma FSH and LH were increased in the PND21/24 BPA rats compared with the control rats (P<0.05). But the levels of plasma T and E₂ were no significant in the PND18/21/24 BPA rats and the control rats (P>0.05). In comparison with control group, the expression levels of POA-GnRH mRNA and ARC-kiss1 mRNA were significantly increased in PND21 BPA rats (GnRH: P<0.05; kiss1: P<0.01). Conclusions: In addition to the direct effect of BPA on the testes of male offspring, the perinatal exposure to a low dose of BPA promotes spermatogenesis via advancing the pubertal activation of ARC-kisspeptin, and via further enhancing the expressions of meiotic regulation genes in the testes of male offspring.

Key words: Spermatogenesis, Peripartum period, Meiosis, Hypothalamus, Animal experimentation, Rats, Bisphenol A

白银阳, 熊芳, 张昀, 陈洁, 徐丽爽, 汪敏. 围生期低剂量双酚A对子代雄鼠生精功能的影响[J]. 国际生殖健康/计划生育, 2021, 40(5): 353-358.

BAI Yin-yang, XIONG Fang, ZHANG Yun, CHEN Jie, XU Li-shuang, WANG Min. The Influence of Perinatal Exposure of Low Dose of Bisphenol A on Spermatogenesis of Male Offspring Rats[J]. Journal of International Reproductive Health/Family Planning, 2021, 40(5): 353-358.

图/表 6

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基因名称	上游引物（5′—3′）	下游引物（5′—3′）	大小（bp）
kiss1	CTTCTCCTCTGTGTGGCCTCTTTTG	GGAGTTCCAGTTGTAGGCTGACATG	312
GnRH	CTGTTGTTCTGTTGACTGTGTGTTTGG	CATCTTCTTCTGCCCAGCTTCCTC	248
cyclin A1	GTTCCAACCACCAACCAGTTCCTC	CTCACTCAGGCAAGGCACAATCTC	255
cyclin B	CACCAAGCACCAGATCAGACAGATG	ACGGTTCACCATGACTATGTTCTTAGC	346
c-jun	CAATGGGCACATCACCACTACACC	TTGAAGTTGCTGAGGTTGGCGTAG	279
c-fos	AAGCGGAGACAGATCAACTTGAAGAC	CATTGAGAAGAGGCAGGGTGAAGG	240
GAPDH	GGTGATGCTGGTGCTGAGTATGTC	CAGTCTTCTGAGTGGCAGTGATGG	301

基因名称	上游引物（5′—3′）	下游引物（5′—3′）	大小（bp）
kiss1	CTTCTCCTCTGTGTGGCCTCTTTTG	GGAGTTCCAGTTGTAGGCTGACATG	312
GnRH	CTGTTGTTCTGTTGACTGTGTGTTTGG	CATCTTCTTCTGCCCAGCTTCCTC	248
cyclin A1	GTTCCAACCACCAACCAGTTCCTC	CTCACTCAGGCAAGGCACAATCTC	255
cyclin B	CACCAAGCACCAGATCAGACAGATG	ACGGTTCACCATGACTATGTTCTTAGC	346
c-jun	CAATGGGCACATCACCACTACACC	TTGAAGTTGCTGAGGTTGGCGTAG	279
c-fos	AAGCGGAGACAGATCAACTTGAAGAC	CATTGAGAAGAGGCAGGGTGAAGG	240
GAPDH	GGTGATGCTGGTGCTGAGTATGTC	CAGTCTTCTGAGTGGCAGTGATGG	301

检测时间	组别	n	生精小管直径（μm）	支持细胞体积（mm³）	精原细胞体积（mm³）	精母细胞体积（mm³）	圆形精子细胞管腔率（%）	圆形精子细胞数量
PND18	对照组	10	91.12±8.55	4.91±0.75	0.41±0.07	0.34±0.06	0	0
	BPA组	10	93.43±7.71	5.39±0.55	0.38±0.06	0.32±0.07	0	0
	t		-0.63	-1.67	1.04	0.75	-	-
	P		0.54	0.11	0.31	0.46	-	-
PND21	对照组	10	108.73±5.59	5.41±0.66	0.44±0.06	0.74±0.08	0	0
	BPA组	10	110.84±6.07	5.98±0.71	0.39±0.08	0.81±0.08	0	0
	t		-0.80	-1.87	1.49	-1.80	-	-
	P		0.43	0.08	0.15	0.09	-	-
PND24	对照组	10	131.03±8.21	7.71±0.73	0.57±0.09	1.31±0.10	10.31±2.67	100.40±11.97
	BPA组	10	140.82±9.34	8.13±0.84	0.50±0.08	1.39±0.11	20.52±3.78	410.50±41.78
	t		-2.49	-1.14	1.69	-1.50	-6.97	-22.55
	P		0.02	0.27	0.11	0.15	0.00	0.00

检测时间	组别	n	生精小管直径（μm）	支持细胞体积（mm³）	精原细胞体积（mm³）	精母细胞体积（mm³）	圆形精子细胞管腔率（%）	圆形精子细胞数量
PND18	对照组	10	91.12±8.55	4.91±0.75	0.41±0.07	0.34±0.06	0	0
	BPA组	10	93.43±7.71	5.39±0.55	0.38±0.06	0.32±0.07	0	0
	t		-0.63	-1.67	1.04	0.75	-	-
	P		0.54	0.11	0.31	0.46	-	-
PND21	对照组	10	108.73±5.59	5.41±0.66	0.44±0.06	0.74±0.08	0	0
	BPA组	10	110.84±6.07	5.98±0.71	0.39±0.08	0.81±0.08	0	0
	t		-0.80	-1.87	1.49	-1.80	-	-
	P		0.43	0.08	0.15	0.09	-	-
PND24	对照组	10	131.03±8.21	7.71±0.73	0.57±0.09	1.31±0.10	10.31±2.67	100.40±11.97
	BPA组	10	140.82±9.34	8.13±0.84	0.50±0.08	1.39±0.11	20.52±3.78	410.50±41.78
	t		-2.49	-1.14	1.69	-1.50	-6.97	-22.55
	P		0.02	0.27	0.11	0.15	0.00	0.00