Gestational Exposure to Bisphenol A Affects Testicular Morphology, Germ Cell Associations, and Functions of Spermatogonial Stem Cells in Male Offspring

Exposure to bisphenol A (BPA) in the gestational period damages the reproductive health of offspring; detailed evidence regarding BPA-induced damage in testicular germ cells of offspring is still limited. In this study, pregnant mice (F0) were gavaged with three BPA doses (50 μg, 5 mg, and 50 mg/kg body weight (bw)/day; tolerable daily intake (TDI), no-observed-adverse-effect-level (NOAEL), and lowest-observed-adverse-effect level (LOAEL), respectively) on embryonic days 7 to 14, followed by investigation of the transgenerational effects of such exposure in male offspring. We observed that the NOAEL- and LOAEL-exposed F1 offspring had abnormalities in anogenital distance, nipple retention, and pubertal onset (days), together with differences in seminiferous epithelial stages and testis morphology. These effects were eradicated in the next F2 and F3 generations. Moreover, there was an alteration in the ratio of germ cell population and the apoptosis rate in germ cells increased in F1 offspring at the LOAEL dose. However, the total number of spermatogonia remained unchanged. Finally, a reduction in the stemness properties of spermatogonial stem cells in F1 offspring was observed upon LOAEL exposure. Therefore, we provide evidence of BPA-induced disruption of physiology and functions in male germ cells during the gestational period. This may lead to several reproductive health issues and infertility in offspring.


Supplementary Methods 19
Collection of testis and determination of testicular abnormalities 20 F1 male mice were selected from each dam and sacrificed at PNDs 30, 60, and 120, following 21 which the testes were collected and weighed. Testes of F2 and F3 males were collected at PND 120.

22
The testes were dissected vertically into two parts: one for paraffin sectioning and the other for 23 fluorescence-activated cell sorting (FACS). Testis parts used for sectioning were fixed in Bouin's 24 solution at room temperature for 6 h and subsequently washed with 70%-100% ethanol gradient to 25 dehydrate the tissue at intervals of 5 min, followed by washing with xylene. Testis tissues were 26 embedded in paraffin wax, and five micrometer-thick serial sections were cut and placed on glass 27 slides. Some of the slides were stained with hematoxylin and eosin for examination of testicular 28 morphology under a microscope (TE2000-U, Nikon, Chiyoda-ku, Tokyo, Japan). Seminiferous 29 tubules (STs) with huge lumen or with no lumen, abnormal cell mass inside the lumen area, and germ 30 cell loss in the seminiferous epithelium (SE) or presence of vacuoles in the SE were considered as 31 testicular abnormalities. The measure of testicular abnormalities was examined according to a 32 procedure previously described by Doyle et al., 2013 [1]. All STs in one section were considered for 33 obtaining data.  The transplantation procedure was conducted according to a previously described protocol [2,4].

53
Most of the effects related to BPA exposure were observed in F1 offspring and at NOAEL and LOAEL 54 doses. Therefore, F1 males exposed to these two doses were selected as germ cell donors for this experiment along with control and positive control (EE-exposed) groups. Six-week-old CD-1 male 56 mice were selected as recipients. Recipient mice were prepared 6 weeks before the day of 57 transplantation by injecting busulfan into the intraperitoneal cavity at a concentration of 35 mg/kg 58 body weight to eradicate endogenous germ cells and spermatogenesis from the recipient testes.

59
Collection of germ cells from donor mice was conducted just before transplantation. Donor mice 60 were sacrificed, their testes were collected, and tunica albuginea from the testis was removed.

61
Testicular germ cells were separated using the same procedure described in the 'Flow cytometric 62 analysis' section. After filtration with a 40-µ m pore sized mesh, the filtered cells were stained using 63 a membrane linker dye (PKH26; red fluorescence, Sigma-Aldrich) at a concentration of 3.2×10 -6 M 64 and by following the manufacturer's protocol. The proportion of SSCs in a mouse testis is ~0.01% of 65 the total testicular cells [5]. Therefore, we prepared a cell suspension at a concentration of 50×10 6 66 cells/mL with a solution containing 10% (v/v) FBS and 10% DNase I (7 mg/mL) dissolved in minimum 67 essential medium α (Gibco). Following that, the recipient mice were anesthetized using 75 mg/kg 68 ketamine and 0.5 mg/kg medetomidine before transplantation. A hair trimmer was used to remove 69 the lower abdominal hair and the area of surgery was disinfected using iodine and ethanol (70%). A 70 small surgical wound was made, and the testis was driven out carefully from the abdomen. The 71 suspension of donor germ cells was then labeled with 7% (v/v) trypan blue and injected into the 72 recipient testes through efferent ducts, as described previously [6]. About 8-10 µ L (5.0×10 5 cells) of 73 donor germ cell suspension was injected into each testis, which filled 80% of the surface 74 seminiferous tubules.

75
One month after transplantation, the recipient mice were euthanized and the collected testes