Necroptosis promotes the Aging of the Male Reproductive System in Mice

Necroptosis is a form of programmed necrotic cell death in mammals that is mediated by a pair of kinases, RIP1 and RIP3, as well as the RIP3 substrate MLKL. We report here that male reproductive organs of both RIP3-and MLKL-knockout mice retain “youthful” morphology and function into advanced age, while those of age-matched wild type mice deteriorate. The RIP3 phosphorylation of MLKL, the activation marker of necroptosis, is detected in spermatogonial stem cells in the testes of old but not in young wild type mice. When the testes of young wild type mice are given a local necroptotic stimulus, their reproductive organs showed accelerated aging. Feeding of wild type mice with an RIP1 inhibitor prior to the normal onset of age-related changes in their reproductive organs blocked the appearance of signs of aging. Thus, necroptosis in testes promotes the aging-associated deterioration of the male reproductive system in mice.


Introduction 22
Necroptosis is a form of programmed necrotic cell death caused by the tumor necrosis 23 pairs of 10-week old wild type female mice. As summarized in Figure 1H, both wild type and 136 RIP3-knockout 4-month old male mice were fully fertile, and both groups sired a similar number 137 of pups (Figure 1-table supplement 1). However, for 13-month-old mice, only 9 of the 20 wild 138 type male mice sired pups, while 18 out of 23 RIP3-knockout males remained fertile ( Figure 1H

RIP3 expression in spermatogonia, spermatocytes and Sertoli cells in testis 150
To investigate the underlining mechanism responsible for the delayed reproductive system 151 aging phenotype, we first examined RIP3 expression using immunohistochemistry methods 152 (IHC). We noted that the cells inside wild type seminiferous tubules were stained positively with 153 anti-RIP3 antibody (Figure 2-figure supplement 1). In contrast, no staining was seen in the 154 seminiferous tubules of RIP3-knockout mice, confirming the specificity of the antibody ( Figure  155 2-figure supplement 1). 156 The specific cell types from testes were further analyzed by co-immunostaining of testis 157 sections from sexually-mature wild type mice (8-weeks) with antibodies against RIP3 and other did not express RIP3 (Figure 2A). The RIP3 expression in each of these cell types was further 163 confirmed when testes were dissected and the cells were spread on a slide and analyzed again 164 with co-immunostaining. The cell shapes changed due to spreading with this method, but the 165 individual cells were more clearly visible. Consistent with the IHC staining results, 166 spermatogonia and Sertoli cells were positive for RIP3 staining while Leydig cells were not 167 ( Figure 2B). Moreover, the primary spermatocytes within seminiferous tubules that were not 168 marked by IHC were now clearly visible when stained with the specific marker SMAD3 169 (Hentrich et al., 2011), and these cells expressed RIP3 ( Figure 2B). The fact that the cells within 170 seminiferous tubules, the sperm-producing unit of testis, are all positive for RIP3 expression 171 raised a possibility that the age-associated depletion of these cells is through necroptosis. 172

173
The RIP3 substrate MLKL is phosphorylated in the seminiferous tubules of aged wild type 174 mice 175 Recall that RIP3 transduces the necroptosis signal by phosphorylating the serine 345 of 176 pesudokinase MLKL, we used an antibody against phospho-serine 345 of MLKL to analyze the 177 testes of young and old mice. Phosphorylated MLKL (phospho-MLKL) was detected in 178 seminiferous tubules in cells surrounding the center lumens in testes of 18-month old wild type 179 mice, whereas no phospho-MLKL was detected in the same tissue area of 8-week old wild type 180 mice, nor in 18-month old RIP3-knockout mice ( Figure 2C). A quantitative analysis of the 9 phospho-MLKL staining of each age and genotype group is shown in Figure 2D. The data 182 clearly showed that necroptosis-activation marker, i.e. serine-345 phosphorylation, was present 183 abundantly in the seminiferous tubules of old wild type mice but not in young and RIP3-184 knockout mice, thus suggesting that necroptosis of these RIP3-expressing cells in seminiferous 185 tubules might trigger the aging of male sex organs. Consistently, phospho-MLKL was detected 186 by western blotting in extracts from testes of 18-and 24-month old wild type mice but not in 187 extracts from age-matched RIP3 knockout mice ( Figure 2E). 188 To further identify the exact cell type in the aged seminiferous tubules that show positive 189 marker of necroptosis, we co-stained the testis sections with antibodies that specifically mark the 190 different cell types in seminiferous tubules. As shown in Figure 2F, spermatogonia that 191 specifically expressing UTF1 were co-stained with the anti-phospho-MLKL antibody. On the 192 other hand, Sertoli cells did not show phosphor-MLKL staining even though they do express 193 RIP3. Not surprisingly, Leydig cells that do not have RIP3 expression also did not stain with the 194 phosphor-MLKL antibody. 195

Activation of apoptosis in Leydig cells during aging 197
The sex hormone-producing Leydig cells in testes do not express RIP3, yet in old mice 198 testis, the hormone level drops and Leydig cells are also gone. We therefore checked the 199 cleavage status of procaspase-3 (a known marker of apoptosis) and procaspase-8 in the aged 200 testes of wild type and RIP3-knockout mice using IHC. Cleaved procaspase-3 and Cleaved 201 procaspase-8 was detected in the wild type Leydig cells of 18, and 36-month old mice, while no 202 such signal was observed in age-matched RIP3-knockout mice (Figure 3A-3D and Figure3-203 figure supplement 1A,1B). The cleaved-Caspase-3 was also detected by western blotting using 204 extracts from the aged wild type testes but not in RIP3-knockout testes (Figure 3-figure  205 supplement 1C). It is thus likely that Leydig cells undergo apoptosis, as a secondary response to 206 necroptosis in seminiferous tubules during aging process. 207 208 Caspase-8 levels decrease during aging in empty seminiferous tubules 209 We also used immunohistochemistry methods to examine the caspase-8 level in relative 210 to RIP3 in testes of wild type mice of advanced age. In aged wild type mice, caspase-8 levels 211 The delayed testis aging phenotype of RIP3 knockout mice and detection of necrptosis 218 activation marker in spermatogonia in aged wild type mice suggest that necroptosis might be the 219 underlying cause of testis aging. To further investigate possibility, we also characterized the 220 aging-associated phenotype of MLKL knockout mice. We first weighed 15-month old wild type, 221 RIP3-knockout, and MLKL-knockout (MLKL -/-) mice. There was no significant difference 222 between the weights of MLKL-and RIP3-knockout mice, and mice of both of these knockout 223 genotypes weighed less than wild type mice at this age ( Figure 4A). We also analyzed seminal 224 vesicles and seminiferous tubules in aged MLKL-knockout mice (15-month old). Compared to 225 the obvious aging that had occurred in wild type mice, the seminal vesicles of MLKL-knockout 226 mice maintained a youthful appearance, exhibiting the same phenotype as RIP3-knockout mice 227 ( Figure 4B). Furthermore, while the majority of seminal vesicles from 15-month old wild type 228 mice weighed more than 1,000 milligrams, almost all of the seminal vesicles from age-matched 229 MLKL-and RIP3-knockout weighed less than 1,000 milligrams ( Figure 4C). Consistently, the 230 testosterone levels of both MLKL-and RIP3-knockout mice were also significant higher than 231 those of age-matched wild type mice ( Figure 4D). Further, very few (<2%) of the seminiferous 232 tubules from MLKL-knockout mice were empty at 15 months of age, similar to the tubules of 233 RIP3-knockout mice, while more than 12% of seminiferous tubules from the age-matched wild 234 type mice were already empty ( Figure 4E, 4F). Finally, the fertility rates of both 16-Month old 235 MLKL-and RIP3-knockout mice were also significant higher than those of age-matched wild 236 type mice ( Figure 4G). 237 238

Induction of necroptosis in testis depleted cells in seminiferous tubules 239
To directly demonstrate that necroptosis in testes is sufficient to cause the aging of the 240 In addition to monitoring these short-term effects following TSZ injection of 3-month-old 257 mice, we waited for three additional months following the injection and assessed the long-term 258 effects of induced necroptosis in mouse testes. Interestingly, three months after TSZ injection, 259 the seminal vesicles of wild type recipient mice were as enlarged as those from mice older than 260 15 months. However, no such enlargement of seminal vesicles was observed in RIP3-and 261 MLKL-knockout mice after the same TSZ treatment of their testes ( Figure 6A, 6B). Additionally, 262 more than 30% of the wild type seminiferous tubules remained empty three months after the 263 injection, while those of RIP3-and MLKL-knockout mice appeared completely normal without 264 any observable loss of cells ( Figure 6C, 6D). 265 We also tested the fertility rate of TSZ-treated mice 3-month after the TSZ treatment. 266 Control injection of saline into the testes of wild type mice did not affect the fertility rate and the 267 mice remained 100% fertile, but TSZ injection reduced the fertility rate by 87.5% (only 1 of 8 268 was fertile) ( Figure 6E). In contrast, 6 out of 8 RIP3-knockout mice and 7 out of 8 MLKL-269 knockout mice were still fertile following TSZ injection ( Figure 6E). We subsequently chose the 300 mg/kg dose to continuously feed 13-month old male wild 282 type mice for two months to study the long-term effects of blocking necroptosis on testes. After  Additionally, the testosterone level of RIPA-56-treated mice remained high, while that of control 288 mice decreased ( Figure 7E). Consistently, more than 12% of the seminiferous tubules of the 289 control mice were empty, whereas hardly any seminiferous vesicles were empty in the RIPA-56-290 treated mice ( Figure 7F, 7G). Finally, the fertility rates of the RIPA-56-treated mice were much 291 higher than those of control mice with 19 out of 25 mice on the RIPA-56 diet were fertile while 292 only 6 out of 23 mice on normal diet produced progeny ( Figure 7H). 293 294

Discussion 295
Necroptosis in testis promotes the aging phenotype of mouse male reproductive system 296 The above presented data indicated that the previously unknown physiological function of 297 necroptosis is to promote the aging of male reproductive organs. We detected for the first time 298 under physiological conditions the activation marker of necroptosis in spermatogonia of old 299 testis. Consistently, mice with either of their core necroptosis execution components RIP3 and 300 MLKL deleted from their genome showed dramatic delay of male reproductive aging phenotype, 301 both morphologically and functionally. 302 The delay of aging phenotype seems to be restricted to the reproductive system. We 303 conducted histological analysis of major organs including small intestines, spleen, lung, liver, 304 large intestines, kidney, heart, and brain of wild type and RIP3-knockout mice aged 8 weeks, 4 305 months, 18 months, and 24 months, and observed no differences between wild type and age-306 matched RIP3-knockout mice during the aging process (Figure 1-figure supplement 6). 307 The fact that one dose of TSZ treatment applied locally to the testes could mimic the aging 308 phenotype, including the enlargement of seminal vesicles, the depletion of cells in the 309 seminiferous tubules, and decreases in fertility rates in wild type but not RIP3-knockout or 310 MLKL-knockout mice, strongly suggests that necroptosis happening within seminiferous tubules 311 is the cause of symptomatic male reproductive system aging. 312 313 Necroptosis-promoted male reproductive system aging offers an evolutionary advantage at 314

species level 315
When we examined the progenies sired by the aged RIP3-knockout mice at a time wild type 316 mice had lost their reproductive activity, we found that they were less healthy than the progenies 317 sired by young males, with higher rates of prenatal and postnatal death ( hormones LH and FSH declined in RIP3-knockout mice as they age just as they do in wild type 325 mice, it is obvious that other age-related changes in DNA in their gametes and other organs 326 occur normally in these mutant mice. Therefore, although mice without the core components of 327 the necroptosis pathway maintain their reproductive activity into advanced ages (well beyond the 328 age when wild type mice have largely lost such capacity), these age-associated, non-necroptotic 329 changes still caused deleterious effects on their progeny. 330 We therefore propose that necroptosis in seminiferous tubules is a physiological response to 331 yet-to-be-identified, age-related, TNF family of cytokine(s) that transduces necroptosis signal 332 through the canonical RIP1-RIP3-MLKL pathway. The necroptotic death of cells in seminiferous 333 tubules of testis then triggers the other downstream age-related phenotypes such as enlargement 334 of seminal vesicles, decreased testosterone levels and weight gain. Given the large number of 335 TNF family of cytokines and the signal seems to only act on spermatogonia, the identification of 336 such a signal is a challenge yet interesting research topic for the future studies. 337 Necroptosis-instigated reproductive system aging effectively eliminates old animals from 338 the reproductive pool. Given that aged animals carry significantly more DNA damage than 339 younger animals, their elimination from the mating pool results in healthier pups on the whole, 340 an outcome that would confer an evolutionary advantage over (a population) of animals that do 341 not thusly employ a necroptosis program in their testes. 342 Interestingly, when wild type mice were fed with food containing an RIP1 inhibitor prior to 343 the onset of reproductive system aging (13 months), the aging of the male reproductive system 344 could be completely blocked. This finding not only further confirms that necroptosis is the 345 mechanism underlying male reproductive system aging, but also demonstrates an apparently-346 effective way to delay it. 347 348

Harvesting of tissues 381
Mice were euthanized using avertin (20 mg ml -1 ). Animals were euthanized one by one 382 immediately before dissection, and the dissection was performed as rapidly as possible by a team 383 of several trained staff members working in concert on a single mouse. Major organs were 384 removed, cut into appropriately-sized pieces, and either flash-frozen in liquid nitrogen and stored 385 at -80°C or placed in formalin (Using Bouin's fixative for testis) for preservation. After several 386 days of formalin fixation at room temperature, tissue fragments were transferred to 70% ethanol 387 and stored at 4°C. Blood was collected by cardiac puncture, and was allowed to coagulate for the 388 preparation of serum. 389

Western blotting 390
Western blotting was performed as previously described (

Mating and fertility test 443
Mice (male) used for the fertility tests were housed individually in an SPF barrier facility. 444 To assess vaginal patency, mice were examined daily from weaning until vaginal opening was 445 observed. The fertility rate of males was determined via a standard method (Cooke and Saunders, 446 2002; Hofmann et al., 2015) by mating a male with a series of pairs of 10-week old wild type 447 females for 3 months; females were replaced every 2 weeks (females were either from our 448 colony or purchased from Vital River Laboratory Co(C57BL/6)). Each litters was assessed from 449 the date of the birth of pups; when pups were born but did not survive, we counted and recorded 450 the number dead pups; for females that did not produce offspring, the number of pups was 451 recorded as '0'(did not produce a litter with a proven breeder male for a period of 2 months). 452 The number of male mice with reproductive capacity was recorded. 453 The reproductive longevity of males was determined by continuously housing 2-month old 454 RIP3 +/+ and RIP3 -/males with a pairs of 10-week old wild type females, the females being 455 replaced every 2 months, until males ceased reproducing (calculated as the age at birth of the 456 litter less 21 days) (Hofmann et al., 2015). 457

Isolation of cells from testes 458
Testes from 8-week old mice were collected using a previously-reported protocol (Chang osc/min. After incubation, 3 mL charcoal-stripped FBS was immediately added to halt the 468 digestion. All fractions were mixed and immediately centrifuged at 500 × g at 4°C for 10 min. 469 Pellets were re-suspended in PBS and washed 3 times, then cultured in DMEM:F12(10%FBS) 470 medium at 37°C. 471

RIPA-56 feeding experiment 472
RIPA-56 in the AIN93G (LAD3001G) at 150 or 300 mg/kg was produced based on the 473 Trophic Animal Feed High-tech Co's protocol. Cohorts of 13-month old wild type male mice 474 were fed with AIN93G or AIN93G containing RIPA-56 (RIPA-56:300mg/kg) for 2 months in an 475 SPF facility; each male mouse was then mated with four 10-week old wild type female mice 476 successively. The number of male mice with reproductive capacity were recorded. 477

Statistical analysis 478
All experiments were repeated at least twice. Data represent biological replicates.