Tubulointerstitial nephritis antigen‐like 1 deficiency alleviates age‐dependent depressed ovulation associated with ovarian collagen deposition in mice

Abstract Purpose This study aimed to examine whether the Tinagl1 might be associated with ovulation in aged females and reproductive age‐associated fibrosis in the stroma of the ovary. Methods To address the ovulatory ability and quality of ovulated oocytes, we induced ovulation by treatment with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) followed by in vitro fertilization. We also performed Picrosirius Red (PSR) staining to evaluate ovarian collagen deposition. Results As compared to ovulation in 8‐ to 9‐month‐old Tinagl1flox/flox mice, the number of ovulated oocytes from Tinagl1flox/flox mice decreased in an age‐dependent manner in mice more than 10‐11 months old, whereas the ovulated oocyte numbers in Tinagl1 −/− mice decreased significantly at 14‐15 months. In vitro fertilization followed by embryo culture demonstrated the normal developmental potential of Tinagl1‐null embryos during the preimplantation period. PSR staining indicated that collagen was found throughout the ovarian stroma in an age‐dependent manner in Tinagl1flox/flox females, whereas those distributions were delayed to 14‐15 months in Tinagl1 −/− females. This timing was consistent with the delayed timing of age‐related decline of ovulation in Tinagl1 −/− females. Conclusions The alleviation of age‐associated depression of ovulation was caused by delayed ovarian collagen deposition in Tinagl1‐null female mice.

In addition, the reduction of gamete quantity is characterized as physiologic aging. While the number of available oocytes is reduced in an age-dependent manner, rare follicles were observed in ovaries, and the ovarian cortex was almost displaced by connective tissue in aged mice. 1 In mice, reproductive age-associated fibrosis occurred in the stroma of the ovary, as evidenced by the observation that Picrosirius Red (PSR) staining, specific for collagen I and III, was minimal in ovaries from reproductively young adult mice (6-12 weeks), increased in distinct foci in mice of midto-advanced reproductive age, and prominent throughout the stroma of the oldest mice in the study (>20 months). 9 Therefore, the distribution of extracellular matrix (ECM) components appears to be involved in age-associated reproductive decline, including ovulation.
In contrast to the ECM proteins such as collagens, which contribute to the structural integrity of the extracellular matrix by multimerization, a group of ECM proteins that do not directly play a structural role is categorized as matricellular proteins. 10 Matricellular proteins interact with structural matrix proteins, cell surface receptors, or extracellular factors such as growth factors and proteinases, and modulate cell-matrix interactions and cellular functions, including proliferation, differentiation, adhesion, and migration.
Furthermore, several matricellular proteins that are associated with collagen deposition are known to be involved in hepatic fibrosis, including osteopontin (OPN), [11][12][13] tenascin C, 14 cysteine-rich acidic secreted protein (SPARC), [15][16][17] and periostin. 18 Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) is a matricellular protein that interacts with both structural matrix proteins and cell surface receptors. [19][20][21] Our previous study revealed that homologous mating of Tinagl1 −/− females and Tinagl1 −/− males showed impaired fertility during pregnancy, including failure to carry pregnancy to term and perinatal lethality. 22 Meanwhile, ovulation from 2-to 7-month-old Tinagl1 −/− mice induced by equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) showed that the number of ovulated oocytes did not differ compared with the number in Tinagl1 flox/flox mice. 22 In vitro fertilization followed by embryo culture also demonstrated normal developmental potential of Tinagl1-null embryos during the preimplantation period. 22 In aged mice, the ovarian cortex was almost displaced by connective tissue. 1 Furthermore, reproductive age-associated fibrosis was apparent in the stroma of the ovary in mice. 9 As Tinagl1 is a matricellular protein, Tinagl1 deficiency could be associated with ovulation in aged females. Our initial expectation in the present study was that age-associated depression of ovulation in aged Tinagl1 −/− females might be slight as compared with Tinagl1 flox/flox females. To address the ovulatory ability and quality of ovulated oocytes, we induced ovulation by treatment with eCG and hCG followed by in vitro fertilization. We also performed PSR staining in ovaries to examine the accumulation of collagen and investigated whether the amount of collagen deposition differed between Tinagl1 −/− and Tinagl1 flox/flox during aging.

| Animals
Tinagl1-deficient mice were generated as described previously. 22 Tinagl1 −/− and Tinagl1 flox/flox mice were maintained by homologous matings. These mice were in the C57BL/6NCr background. Genotype was determined by PCR analysis of genomic DNA. Mice were bred in our animal care facility. The animal experiments described here were approved by the Animal Experimentation Committee at the Utsunomiya University and were performed in accordance with the instructions in the Guide for the Care and Use of Laboratory Animals published by Utsunomiya University.

| Superovulation, in vitro fertilization, and embryo culture
Superovulation, in vitro fertilization, and culture of embryos were performed as previously described. 22 overlaid with paraffin liquid (Nacalai Tesque) and cultured in a humidified atmosphere with 5% CO 2 at 37°C.

| Picrosirius Red (PSR) staining
Picrosirius Red staining was performed as previously described 9,24,25 with slight modifications. Ovarian tissues were snap frozen following collection of ovulated oocytes using freezing solution (FREEZER, HOZAN) as described previously. 20 and mounted with mounting medium (Daido Sangyo Co.). ImageJ software (National Institutes of Health) was used to process color threshold images of PSR-stained ovarian tissue sections as described previously. 9 Three mice were used for each genotype and age group.

| Immunohistochemical staining of Tinagl1
Immunohistochemistry was performed as described previously. [20][21][22] Frozen 10-µm sections from snap-frozen tissues were mounted onto silane-coated glass slides. Sections were fixed in cold acetone on ice, immersed in 3% hydrogen peroxide in methanol, and treated with 1% BSA in PBS. For the primary antibody reaction, sections were incubated with rabbit polyclonal antibody to Tinagl1. After washing, sections were incubated with biotinylated goat anti-rabbit antibody (Thermo Fisher Scientific, Inc). After incubation with horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Inc), reactions were visualized using 3-amino-9-ethyl carbazole (Thermo Fisher Scientific, Inc) as a chromogen, followed by counterstaining with hematoxylin. Reddish deposits indicate the sites of immunoreaction.

| Development to blastocyst is not affected by Tinagl1-deficiency
As described above, the fertilization potential of ovulated oocytes did not differ significantly between Tinagl1 flox/flox and Tinagl1 -/mice.
To address the further developmental ability, embryo culture was employed to determine the preimplantation development to the blastocyst stage. Development to the blastocyst stage or beyond was used as a parameter for successful in vitro development during the preimplantation period. The scatterplot in Figure 3A  In contrast, for Tinagl1 −/− females, r was −0.23, and there was no F I G U R E 2 Effect of female aging on cleavage of fertilized eggs derived from Tinagl1 flox/flox and Tinagl1 −/− mice to the 2-cell stage or beyond. A, Scatterplots displaying the correlation between age and percentages of fertilized eggs. B, Bimonthly profile of the percentages of fertilized eggs derived from Tinagl1 flox/flox females. The same letter (a above error bars) indicates no significant differences among the age groups (P > .05). C, Bimonthly profile of the percentages of fertilized eggs derived from Tinagl1 −/− females. The same letter (a above error bars) indicates no significant differences among the age groups (P > .05). D, Comparisons of the percentages of fertilized eggs between Tinagl1 flox/flox and Tinagl1 −/− mice in the same age groups. The same letter (a above error bars) indicates no significant differences between  Figure 3B. The percentages (mean ± SEM) of blastocysts from 8-to 9-, 10-to 11-, 12-to 13-and 14-to 15-months-old females were 77.3 ± 3.3 (n = 11), 77.3 ± 6.4 (n = 14), 61.9 ± 6.6 (n = 11), and 61.6 ± 9.8 (n = 10), respectively. The percentages of blastocysts were not significantly different among the age groups (P > .05). The percentages of blastocysts from Tinagl1 −/− mice in the different age groups are shown in Figure 3C. The percentages of blastocysts (mean ± SEM) from 8-to 9-, 10-to 11-, 12-to 13-, and 14-to 15-month-old females were 81.6 ± 3.1 (n = 15), 70.7 ± 4.0 (n = 22), 65.3 ± 4.6 (n = 20), and 64.8 ± 8.6 (n = 15), respectively. There were no significant differences among these groups (P > .05). Figure 3D shows the percentages of blastocysts in the same age groups from mice with different genotypes. The percentages of blastocysts did not differ significantly between Tinagl1 flox/flox and Tinagl1 −/− females for any of the age groups (P > .05). These results indicate that the developmental potential of ovulated oocytes to the blastocyst stage did not differ between the Tinagl1 flox/flox and the Tinagl1 −/− mice.

| Ovarian deposition of collagen is retarded by Tinagl1-deficiency
As shown in Figure 1, Tinagl1-deficient female mice exhibited alleviation of age-related ovulation decline. Therefore, we hypothesized that reproductive age-associated deposition of collagen may take place in the stroma of the ovary during aging and that the age-associated deposition of collagen also may be delayed in Tinagl1 −/− as com-

| Distribution of Tinagl1 in ovary during aging
Immunohistochemistry was performed to determine the localization of Tinagl1 expression in Tinagl1 flox/flox female ovary during aging.
Tinagl1 protein was expressed in ovarian stroma, blood vessels, theca cells, and corpus lutea ( Figure 5). In ovarian stroma, Tinagl1 expression was similar during aging.

| D ISCUSS I ON
Previous work by our group indicated that induced ovulation in 2-7 months old Tinagl1 −/− mice did not differ compared with Tinagl1 flox/flox mice. 22 Furthermore, ovulated Tinagl1-null oocytes possessed developmental potential during the preimplantation period. 22 In contrast, our present study shows that deficiency of Tinagl1 alleviates the age-related decline of ovulation, whereas in vitro fertilization followed by embryo culture for ovulated oocytes showed a normal developmental potential for Tinagl1 −/− embryos during the preimplantation period as compared with Tinagl1 flox/flox embryos.
We found that Tinagl1-deficient female mice exhibit alleviation of age-related ovulation decline. As compared with 8-to 9-monthold mice, the number of ovulated oocytes from Tinagl1 flox/flox mice decreased in an age-dependent manner for females more than 10-11 months old, whereas ovulated oocyte numbers for 8-to 9month-old Tinagl1 −/− mice did not differ significantly from those for 10-to 11-and 12-to 13-month-old mice but decreased significantly in 14-to 15-month-old females. PSR staining intensity, indicating collagen deposition, increased in an age-dependent manner throughout the ovarian stroma in Tinagl1 flox/flox females. Meanwhile, the observation of intense PSR-stained collagen was delayed to 14-15 months in Tinagl1 −/− females. This timing was consistent with the retarded timing of age-related decline of ovulation in Tinagl1 −/− females. Furthermore, Tinagl1 protein was expressed in ovarian stroma during aging. In mice, reproductive age-associated fibrosis occurs in the stroma of the ovary. Previous study revealed that PSR staining was minimal in ovaries from reproductively young adult mice and increased in advanced reproductive age, with staining becoming prominent throughout the stroma of aged mice. 9 Therefore, our current findings suggest that Tinagl1 deficiency causes delayed reproductive age-associated ovarian collagen deposition followed by an alleviation of the age-related decline of ovulation.
Fibrosis occurred in the ovarian stroma with increasing age and increased the inflammatory response in the ovary. 9 TINAGL1 level was increased in Fabry disease with upregulated inflammation-related pathways. 27 Therefore, ovarian expression Tinagl1 may be associated with inflammatory response during ovarian fibrosis. In addition, stiffness of ovary was increased with ovarian stromal fibrosis 28 and showed that the stiffness around the follicle was associated with follicular development. 29 Ovarian stromal fibrosis caused the abnormal follicular development at secondary follicle in granulosa cell-specific Nrg1 knockout mice and resulted in decreased number of ovulated oocytes. 30 It has been reported that tissue stiffness and mammographic density are associated with the risk of breast carcinoma development, suggesting that matricellular proteins may influence breast carcinoma prognosis. 31 Moreover, Tinagl1 suppresses triple-negative breast cancer progression and metastasis. 32 Therefore, ovarian Tinagl1 may be associated with ovarian stiffness and depressed follicular development. Recent study reported that TINAGL1 promotes hepatocellular carcinogenesis through the activation of TGF-β signaling medicated VEGF expression. 33 Because Tinagl1 is present in blood vessels, 19,21,34 Tinagl1 also could be related to ovarian blood vessel formation followed by constant cycles of connective tissue remodeling.
Through the processes of ovulation, the ovary undergoes constant cycles of connective tissue remodeling and wound healing, which require a complex interplay between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). 35 TIMPs inhibit the proteolytic activity of MMPs. Therefore, age-associated changes in the homeostasis of ECM may be associated with an imbalance in the activities of MMPs and TIMPs. 9 Furthermore, the age-associated fibrosis could be due to increased synthesis and deposition of collagen or other ECM components and/or altered post-translational modifications, because α-smooth muscle actin (α-SMA), which corresponds to a myofibroblast population, 36,37 has been observed in the ovarian stroma. 9 Persistent activation of myofibroblasts can cause excessive fibrotic reactions. 38 In addition, the F I G U R E 5 Distribution of Tinagl1 in Tinagl1 flox/flox mouse ovary during aging. The scale bar represents 200 µm study of myofibroblast recruitment and activation in skin wounds using α-SMA-GFP transgenic mice demonstrated that Tinagl1 is linked to myofibroblast-mediated wound healing. 38 Therefore, it is possible that the alleviation of age-associated decline of ovulation in Tinagl1-deficient females is caused by reduced connective tissue remodeling during the processes of ovulation.
Under normal physiologic conditions, tissue remodeling in response to injury leads to tissue regeneration without permanent damage. Matricellular proteins are often upregulated after tissue injury 39 to provide extracellular matrix signals critical for tissue repair processes. 40,41 These non-structural ECM proteins are important during embryonic development but are typically restricted to tissue remodeling and wound repair in normal adults. [42][43][44] Indeed, several studies examining mice deficient in matricellular proteins have identified previously unsuspected consequences stemming from the lack of appropriate interactions between cells and their environment. 10 Tinagl1 has been identified as a matricellular protein. 19 In this work, we determined that Tinagl1 plays a role in age-associated ovulation decline caused by ovarian collagen deposition.
In conclusion, the present study describes new observations including alleviation of age-associated depression of ovulation caused by retarded ovarian collagen deposition in Tinagl1-null female mice. Although the numbers of ovulated oocytes from Tinagl1 flox/ flox mice decreased in an age-dependent manner in mice more than 10-11 months old, ovulated oocyte numbers from Tinagl1 −/− mice did not differ significantly among 8-9, 10-to 11-, and 12-to 13-monthold females and decreased significantly by 14-15 months. However, the fertilization potential of ovulated oocytes was not significantly different between Tinagl1 flox/flox and Tinagl1 −/− mice. Furthermore, Tinagl1 deficiency did not affect the potential of development to the blastocyst stage of ovulated oocytes during the preimplantation period as compared with Tinagl1 flox/flox females. As Tinagl1 deficiency alleviated the age-related decline of ovulation, and in vitro fertilization followed by embryo culture for ovulated oocytes showed normal developmental potential during the preimplantation period, investigation of physiologic and/or microenvironmental conditions in the ovary may further elucidate the role of Tinagl1. Such work could yield information that may help to improve assisted reproductive technology, although determination of the definitive role of Tinagl1 in ovary during aging will require further studies involving the ovary-specific deletion of this gene.

ACK N OWLED G EM ENTS
We wish to thank Eisaku Takeuchi, Saori Suzuki, Daiki Nakashima, and Akihito Takahashi (Utsunomiya University) for their expert technical assistance. This work was supported in part by JSPS KAKENHI Grant Numbers 22580316, 25450390 and 18K05936 (H.M).

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

A N I M A L S TU DY
All animal experiments described here were approved by the Animal Experimentation Committee at the Utsunomiya University and were performed in accordance with the instructions in the Guide for the Care and Use of Laboratory Animals published by Utsunomiya University.

H U M A N R I G HTS
This article does not contain any studies with human subjects performed by the any of the authors.