TMEM150B is dispensable for oocyte maturation and female fertility in mouse

Premature ovarian insufficiency (POI) refers to severe decline of ovary function in females which usually leads to infertility. It has been reported that the TMEM150B gene is mostly associated with age at natural menopause, early menopause and POI, but its role in female reproduction remains unknown. In this study, we found Tmem150b was highly expressed in mouse oocytes, but its deletion had no obvious effect on meiotic maturation of oocytes indicated by first polar body emission and spindle morphology. There were also no obvious differences in follicle development and corpus luteum formation between knockout and wild type mice. Finally, knockout of Tmem150b did not affect female fertility and sexual hormone levels. In summary, our results suggest that TMEM150B is not essential for female fertility in mice.


Determination of hormonal profile and histological analysis.
To detect sex hormone levels from serum, blood was collected through ophthalmectomy immediately after the general anesthetic. According to standard protocol, serum samples were separated by centrifuging at 4 °C, 3000×g for 30 min. Follicle-stimulating hormone (FSH) and estradiol (E 2 ) concentrations were measured by radioimmunoassay (Beijing North Institute of Biotechnology, China). Ovaries from 3-month and 8-month-old mice were collected and fixed in Bouin's solution at 4℃ overnight before being transferred to 70% (vol/vol) alcohol. Ovaries were processed, embedded in paraffin with standard protocols and serially sectioned at 5 μm. Following deparaffinization, ovarian sections were stained with H&E for further histological analysis.
Fertility test. For fertility test, 2-month-old Tmem150b +/+ and Tmem150b -/females were mated with WT males over a period of 6 months. The number of pups was counted at the day of birth. Fertility was assessed by the total number of pups per genotype, average number of litters per mouse, average number of pups per litter and time to first litter. Statistical analysis. Data were presented as mean ± SD and analyzed by Student's t test. Statistical analysis was performed with SPSS 16.0 software. Differences were considered to be statistically significant when P < 0.05.

Results
Oocyte meiotic maturation and spindle organization. We firstly analyzed Tmem150b expression in a variety of mouse tissues by qRT-PCR. Tmem150b mRNA level was low in diverse mouse tissues containing spleen, lung, kidney, testis and ovary, while the expression of Tmem150b was relatively high in oocytes at GV and MII stages (Fig. 1). These results suggested that Tmem150b might play a role in mouse oocytes.
To investigate the potential role of TMEM150B, we generated Tmem150b KO mouse model by deleting exons 2 to 4 ( Fig. 2A). While PCR results using primers (F + R) for wild type allele yielded 1820 bp fragment, knockout allele produced 831 bp fragment. Meanwhile, PCR using another pair of primers (dF + R) yielded 413 bp and no bands for wild type and knockout alleles, respectively (Fig. 2B). We further carried out RT-PCR to validate the disruption of the expression of Tmem150b in ovaries from adult mice (Fig. 2C). While the WT allele yielded 524 bp band, KO allele produced 268 bp band. Sanger sequencing also confirmed the deletion of targeted exons 2 to 4 (Fig. 2D). There were no significant differences in body weight and body length between Tmem150b +/+ and Tmem150b −/− mice according to phenotypical evaluation. MII stage oocytes were collected from WT and KO mice after overstimulation to determine the role of TMEM150B in oocyte meiotic maturation. Similar rates of first polar body (PB1) extrusion were observed in both genotypes (Fig. 3), suggesting that TMEM150B is dispensable for oocyte maturation. www.nature.com/scientificreports/ Next, the spindle morphology of MII stage oocytes was observed from both groups. As shown by immunofluorescence staining, the spindle morphology from Tmem150b -/oocyte was similar to wild type oocyte (Fig. 3A). Furthermore, most metaphase oocytes presented typical fusiform-shaped spindles and well-aligned chromosomes at the equatorial plate, suggesting that absence of TMEM150B has no effect on spindle organization.

Estrous cycle and follicular development of adult mice.
As there were no changes in oocyte maturation at puberty of mice, we wondered whether TMEM150B deletion had any effect on adult female mice. We examined the estrous cycle through vaginal smear with H&E staining every morning for 2 weeks. All the Tmem150b -/mice have regular estrous cycles and normal cell morphology compared with control mice, suggesting that loss of TMEM150B had no obvious effect on estrous cycle (Fig. 4A).
We also observed normal morphology of follicles at different developmental stages from both groups, including primordial follicles, primary follicles, secondary follicles, antral follicles and corpus luteum (Fig. 4B). Granulosa cells were also arranged regularly around oocytes. Collectively, these results suggest that Tmem150b is dispensable for folliculogenesis.

Hormonal profile and female fertility. Fertility test was then performed by mating Tmem150b +/+ and
Tmem150b -/females with WT males for a period of 6 months. Interestingly, all of the adult Tmem150b -/mice were showed normal fecundity during this time duration. As shown in Table1, the total number of pups in Tmem150b -/group was similar to that in control group. In addition, the average numbers of pups per mouse or per litter were also comparable between the two genotypes. Neither the giving birth at the first time showed any differences. All the newborn pups showed similar size between both genotypes.
In addition, we determined whether Tmem150b deletion causes any change in hormonal profile and ovarian morphology in mice aged 8 months. The levels of FSH and E 2 exhibited no obvious alterations between Tmem150b +/+ and Tmem150b -/mice (Fig. 5A). Finally, ovaries from both genotypes showed similar amount of follicles from different stages and corpus lutea (Fig. 5B), suggesting that the Tmem150b -/mice also own regular ovulatory ability.

Discussion
Women suffering from POI face infertility with adverse health outcomes 1 . Despite the etiology of POI has been discovered over decades, the mechanisms still have not been fully investigated. It has been reported that early menopause shares a similar genetic pattern with idiopathic POI 20 . TMEM150B has been recognized as one of candidate genes which are significantly associated with ANM and early menopause 7,8 . Moreover, the SNP rs11668344 located in intron 2 of the TMEM150B gene has been verified to be most significantly associated with POI in European ancestry through genome-wide association study 16 . However, this variant was not verified and no mutation of TMEM150B was identified in the Chinese POI population in our previous study 21 .
It has been reported that the TMEM150B gene was expressed in human ovary 17 . In the present study, we further confirmed the high expression of Tmem150b in mouse oocytes in immature and mature stages. The deletion of genomic DNA from our gene-edited mice was large enough to truncate the TMEM150B protein and make it non-functional. Consequently, we found that the deletion of Tmem150b had no effect on follicle maturation and oocyte development, as indicated by similar PB1 extrusion rate and MII oocyte morphology. Furthermore, www.nature.com/scientificreports/ fertility test showed similar number of pups and litter size in both genotypes. Estrous cycle and hormonal profile also exhibited no significant differences between both genotypes. Our findings suggest that TMEM150B is dispensable for folliculogenesis, oocyte maturation and female fertility in mice. Although our study demonstrates that Tmem150b does not play a prominent role in follicle development and fertility of female mice, there may be some limitations. TMEM150B, also named as DRAM-related/associated member 3 (DRAM-3), is one of the five members of the DRAM family which includes DRAM-1, DRAM-2 and TMEM150A/B/C 17,22 and functional redundancy cannot be excluded. It may be better to mutate all interrelated genes to eliminate the compensation from paralogs. Besides, we only detected the phenotype at basal status in normal condition, and could not exclude the occurrence of POI phenotype under stress conditions.
In summary, we demonstrate that the female Tmem150b -/mice have normal fertility. Together with our previous study identifying no causative variants in POI patients, it is suggested that the TMEM150B gene may not be a POI causative gene. Nevertheless, our study may help the scientific community reveal the etiology of POI.