Spectacular alterations in the female reproductive system during the ovarian cycle and adaptations for matrotrophy in chernetid pseudoscorpions (Pseudoscorpiones: Chernetidae)

Pseudoscorpions are small matrotrophic chelicerates. The embryos develop in a brood sac and feed on the nutritive fluid provided by the female. It was widely accepted that the nutritive fluid is synthesized in the ovary. Recent studies have shown that in Chelifer cancroides, a representative of Cheliferidae, considered one of the most derived pseudoscorpion families, the nutritive fluid is produced not only in the ovary but also in the oviducts. Since evolution of adaptations for matrotrophy in pseudoscorpions is poorly known, we aimed to verify our hypothesis that pseudoscorpions of the family Chernetidae, closely related to Cheliferidae, share the traits of adaptations to matrotrophy in the structure and function of the female reproductive system with C. cancroides. We analysed the structure of the ovary and oviducts in five representatives of chernetids with light, confocal, and transmission electron microscopy. The results confirmed our hypothesis and provided new data which broaden our knowledge of matrotrophic pseudoscorpions. We show that in chernetids, the ovary and oviducts undergo significant alterations including their size, multistep hypertrophy and polyploidization of the epithelial cells involved in secretion of the nutritive fluid, the complex secretory activity of the epithelial cells, massive degeneration of the epithelial cells that have completed secretion, and epithelium renewal.

www.nature.com/scientificreports/ Although the general structure of the ovaries in the species is similar, there are also some notable differences. First, the number of oocytes in the most advanced stages of oogenesis, corresponding to the number of laid eggs, varies from circa 20 in Chernes hahnii (Fig. 1B) and Lamprochernes sp. (not shown), 30 in Allochernes wideri (Fig. 1A), to 40 in Pselaphochernes scorpioides (Fig. 1D) and Pselaphochernes lacertosus (Fig. 1C). Second, in some cases, the ovary is anteriorly bifurcated. In Pselaphochernes lacertosus (Fig. 7I), the anterior branches show a typical ovary structure and contain a similar number of growing oocytes compared to the unbranched part. In Chernes hahnii (Figs. 1B and 7F) and Allochernes wideri (Fig. 7A), the anterior bifurcation is not so evident since only single oocytes or post-ovulatory stalks are occasionally observed on the surface of regions that correspond to the oviducts or ovary branches.
Early and mid oogenic stages. During the early and mid-stages of the oogenic phase, the oocytes that have bulged on the ovary surface undergo previtellogenic growth. The volume of the oocytes is relatively small. The germinal vesicles occupy the cell centre ( Fig. 2A), and the oocytes cytoplasm (ooplasm) is filled with a growing number of organelles. The organelles gather in groups that are clearly visible in the light microscopy. Among the accumulated organelles first lipid droplets appear. Follicular cells are unevenly distributed on the surface of growing oocytes. The oocyte stalks are built of a few relatively small roughly cuboidal cells, grouped at the proximal pole (close to the ovarian wall) of the oocytes (Figs. 2A and 5A). , and Texas-Red-X Agglutinin (red fluorescence). ap, apical part of epithelial cell; epOo, early previtellogenic oocyte; fc, follicular cell; L, lumen; ld, lipid droplet; N, oocyte nucleus; nf, nutritive fluid; od, oviduct; ow, ovarian wall; pn, polyploid nucleus; pOo, previtellogenic oocyte; ps, post-ovulatory stalk; s, stalk; sgd, dark secretory granules; sgl, light secretory granules.  Late oogenic stage (before ovulation). Close to ovulation, the oocytes enlarge during vitellogenic growth. The ooplasm is loaded with a significant number of lipid droplets, and most of them have a great diameter. Among the lipid droplets, very few small dark stained granules, the yolk spheres, are distributed (Fig. 2C). The germinal vesicle is shifted to the peripheral ooplasm (Fig. 2C), which suggests that the oocytes are close to reaching their maturity and in a short time would be ready for ovulation. The stalk cells of vitellogenic oocytes, epithelial cells of the ovary and the oviducts became hypertrophic and polyploid (Figs. 2C, 6A-C and 5C). As a consequence, the shape and size of cells change from small cuboidal to high columnar (Fig. 2C,D). The cytoplasm of pre-ovulatory stalk cells is rich in organelles such as ER cisterns, Golgi complexes, mitochondria, and small secretory vesicles (Figs. 2D and 8B). The secretory vesicles gather in the apical part of the cell, and the cytoplasm of the basal part is occupied by large polyploid and elongated nuclei (Fig. 8B). The stalks of preovulatory oocytes are long and wide with a length comparable to the diameter of vitellogenic oocytes. In the stalks, a narrow lumen appears and the stalks become open (Figs. 2C,D and 5C). ). The length of the postovulatory stalks is many times larger compared to the oogenic phase, which is clearly noticeable when the size of the postovulatory stalks is compared to the stalks of the oocytes arrested in early previtellogenesis (Fig. 3D,E). The morphology of the postovulatory stalk cells, the epithelial cells of the ovarian wall, and the oviduct is alike. They continue hypertrophic growth and polyploidization, and eventually become huge and highly columnar (Figs. 3B,C and 5D). Their polyploid nuclei reach the largest size and maintain the basal position, while their cytoplasm is densely filled with granules (Figs. 3B,C and 5D). The granules are large and represent two categories. One of them, which predominate in the cytoplasm, are intensely stained with methylene blue, whereas, the other, less numerous, show weaker coloration (Fig. 3B,C). In this study, the content of heterogeneous granules was not analyzed with histochemical methods. Among the granules, small lipid droplets are distributed ( Fig. 3C upper inset). Hypertrophic epithelial cells start releasing their content to the lumina of the ovary and postovulatory stalks, and the oviduct (not shown) by detaching small fragments from their apical parts (Fig. 3C lower inset). At this stage of the ovarian cycle, the embryos located in the brood sac are in the early stages of development, that is, before the development of the pumping organ (Fig. 7N). The youngest oocytes in the early meiotic stages change their position compared to the oogenic stage and are located externally to the hypertrophic and polyploid ovarian epithelial cells. In their close neighbourhood small somatic cells are discernible (Fig. 3B). The latter cells do not show any characteristic of hypertrophy or polyploidy. Oocytes arrested in previtellogenesis remain bulged to the body cavity on their short stalks, so close to the ovarian tube (Fig. 3B,D,E).
Advanced secretory phase (nutritive fluid secretion). In the subsequent stage, secretion of the nutritive fluid starts. At first, the fluid appears in the lumen of the postovulatory stalks. The lumen of stalks widens, and the height of the stalk cells becomes reduced, but their cytoplasm still contains numerous secretory granules (Fig. 3F). In the next stage of the secretory phase, the stalks undergo the final increase. They become significantly distended, resembling huge balloons (Figs. 4A-G, 7C,G,L and 5E). The height of epithelial cells that built the . At that stage, the embryos located in the brood sac have developed the pumping organ (Fig. 7O), so they are ready to absorb the nutritive fluid.

Late secretory stage (regeneration).
Once the secretion of the nutritive fluid has been completed and the fluid has left the ovary and the oviduct, the structure of both organs of the female reproductive system changes dramatically and exhibits traits of massive cell degeneration (Fig. 7D,H,M). The embryos developing in the brood sac have already absorbed the nutritive fluid, which is visible inside the embryo's body, in the lumen of a digestive system (Fig. 7P). At this stage, polyploid epithelial cells of the ovarian and oviduct walls degenerate and delaminate from the epithelium. Their cell membranes disintegrate, and polyploid nuclei and remnants of cytoplasm appear in the lumen of the ovary and oviduct (Fig. 9A-D). The place of degenerated cells is taken by a new generation of small non-polyploid epithelial cells. During this time mitotically dividing somatic cells are observed in the ovary (Figs. 9C and 5F) and the oviducts (not shown) which strongly suggests that the epithelium renewal occurs due to mitotic divisions of the somatic cells that have not underwent hypertrophy and polyploidization. The postovulatory stalks shrink and stay attached to the external surface of the ovary wall surrounded by a highly folded basal lamina. The cytoplasm of those stalk cells remains filled with numerous lipid droplets (Fig. 9A,C). During the epithelium regeneration, a part of the stalk cells' content is released to the lumen of the ovary (Fig. 9A arrow). In consecutive stages of the ovarian cycle, the regressed post-ovulatory stalks diminish and become easily distinguishable from the regressed post-ovulatory stalks of the next ovarian cycle ( Fig. 10A and inset).

Discussion
In all investigated species from four genera of the family Chernetidae, the structure of the ovary is similar and typical of chelicerates with growing oocytes exposed to the body cavity. As in other pseudoscorpions, and different from most chelicerates, growing oocytes are enclosed by follicular cells. Previous studies showed that the follicular cells originate from interstitial cells located in the germarium among the youngest germline cells 31 . In chernetids, the arrangement of follicular cells on the oocyte surface is similar to that described in pseudoscorpions from the family Cheliferidae 31 and Cheiridiidae 27 . It strengthens our previous hypothesis that this is a common feature of all pseudoscorpions. In small body-sized arachnids such as schizomids 33 , some mites [34][35][36][37] , and pseudoscorpions 16 , the ovary is unpaired. In much bigger chelicerates, such as xiphosurans 28,38 , scorpions 23,39-41 , solifuges 42 , amblypigids 43 , and spiders 44 , the ovary is either branched or paired. Reduction of the number of ovarian tubes is considered one of the adaptations to obtain a small or miniature body size 26 .
Pioneering studies on ovary structure in representatives of Garypidae 19,21 and Chernetidae 16 revealed that during the secretory phase of the ovarian cycle, the ovary becomes structurally modified. The oocytes growth stops in early previtellogenesis, the ovarian wall thickens, and columnar epithelial cells of the ovarian wall secrete the nutritive fluid. Our previous investigations 31 showed that in C. cancroides, during the secretory phase, the thickening of the ovarian wall is accompanied by the hypertrophy of the stalk cells that do not degenerate after ovulation. Due to this hypertrophy, the postovulatory stalks become the most voluminous structures in the ovary. Our quite recent findings 32 revealed that in Chelifer two organs of the female reproductive system, that is, the ovary and the oviducts, are involved in the synthesis of the nutritive fluid. We also showed that during the secretory phase of the ovarian cycle, stalk cells together with epithelial wall cells of the ovary and oviducts undergo multistep hypertrophy and polyploidization and show structural similarities of secretory active cells 32 . In this study, we demonstrate that in chernetids the ovary and the oviducts undergo structural alterations during consecutive phases of the ovarian cycle, which strongly resemble those described in Chelifer. Those changes include an outstanding increase in the size of the organs of the female reproductive system in the secretory phase of the ovarian cycle caused by high-level hypertrophy and polyploidization of the epithelial cells of the ovarian wall, the stalk cells, and the epithelial cells of the oviducts.
Significantly, the most spectacular growth concerns the oocyte stalks which in the late secretory phase extremely enlarge and look like huge balloons filled with homogenous fluid. So far, the occurrence of such huge stalks has never been reported. There is a possibility, that these structures are unique for some pseudoscorpions including chernetids. On the other hand, the lack of such data in the literature could be explained by the fact that during the secretory phase the pseudoscorpion females occupy their nests, so finding the hidden females is quite difficult. In consequence, this exact phase of the ovarian cycle could be easily omitted for investigations. The latter idea seems to be supported by incomplete investigations conducted by Weygoldt 16,45 on Pselaphochernes scorpioides, which lack a description of the postovulatory stalks. A similar case might probably concern our previous analyses on the ovary structure in C. cancroides 32 .
Previous comparative studies indicate that pseudoscorpions from different families show differences in embryonic development in the amount of reserve materials deposited in the oocytes, the longevity and time of feeding the embryos, and the rate of nutritive fluid production. For example, representatives of chernetids and cheliferids pump the nutritive fluid very early and rapidly, as soon as the pumping organ is functional, while chthoniids pump late and for a long period of time 16 . Some of those differences seem to be closely correlated with the efficiency of nutritive fluid production. As already mentioned, in chernetids, similar to C. cancroides 32 , the www.nature.com/scientificreports/ epithelial cells that produce the nutritive fluid are numerous and represented by three cell populations: (i) the postovulatory stalk cells, (ii) the epithelial ovarian wall cells, and (iii) the epithelial oviductal cells. Otherwise, all mentioned cells are hypertrophic and highly polyploid. It is worth mentioning that hypertrophy of the secretory cells involved in the synthesis of nutrients is quite common among matrotrophic invertebrates, e.g., bryozoans, kamptozoans, bivalves, synascidians and branchiopod crustaceans 1,46 . Polyploidization of secretory epithelial cells is another factor that increases the efficiency of the nutritive fluid synthesis. In chernetids, the amount of nutrients provided by the female for developing embryos seems to be substantial in relation to the small amount of proteid yolk accumulated in the oocytes during vitellogenesis. The high amount of synthesized nutrients also meets the requirements of embryos that share nutrients transferred to the brood sac. It should be reminded here that in chernetids, like in Chelifer, the number of growing oocytes and in consequence developing embryos is quite high and amounts to several dozens ( 16 this study). So, it becomes clear that chernetids in terms of the efficiency of the nutritive fluid production show the same strategy as that known in Chelifer 32 . In pseudoscorpions the time of provisioning the embryos with nutrients is diversified. In chernetids and cheliferids it finishes quite early 16 . Subsequently, the secretion of nutrients is stopped and the next round of the ovarian cycle restarts. Before entering the next stage of the cycle, both organs responsible for the secretion of nutrients undergo additional structural modifications. Although previous studies clearly indicated that the ovary structure is modified during the transition from one ovarian cycle to another it was not evident how does it really happen. Chamberlin in his book 24 only mentioned about rapid degeneration of the ovary which coincides with the extrusion of the egg mass. From Weygoldt's 16 studies on P. scorpioides, it was known that the epithelial cells of the ovary after secretion reduce their height. Makioka stated that in Anchigarypus japonicus the ovarian epithelial cells that have completed the secretion degenerate 19 . Unfortunately, he neither described epithelium degeneration nor referred to the source of stem cells for epithelium renewal. Our previous study on Chelifer 32 , assumed that secretory cells after the secretory phase end with break up, but we were unable to prove this.
In this study, we show that at the end of the secretory phase in the ovary and oviducts of chernetids the vast majority of polyploid epithelial cells involved in secretion degenerate. Degeneration does not include postovulatory stalk cells that shrink and remain on the surface of the ovary during consecutive stages of the ovarian cycle, indicating the number of previously ovulated oocytes. The elimination of a large number of epithelial cells seems to be related to their previous polyploidization. Despite that polyploidization of epithelial cells undoubtedly favours the efficiency of nutrient secretion, it also makes, that epithelial cells after fulfilling their secretory role become transiently redundant until the next secretory phase of the ovarian cycle. Before the next oogenic stage, the ovarian and oviductal epithelium is renewed due to mitotic activity of the cells that have remained unchanged by polyploidization and hypertrophy. The best candidates for stem cells in epithelium renewal are the somatic cells that remain on the surface of the ovarian wall in the close vicinity to the early previtellogenic oocytes. These somatic cells are at early stages of differentiation, and their descendants that appear in the ovary follow the same pattern of development as their ancestors and initially differentiate into the stalk cells of oocytes, which restart previtellogenic growth, and rebuild the wall of the ovary and the oviduct. Eventually, in the next secretory phase, they undergo hypertrophy, polyploidization, etc.
To our knowledge, this is the first report showing the renewal of the epithelium in the female reproductive system of pseudoscorpions. It is worth underlying that degeneration of somatic cells and in consequence the epithelium renewal occur on a great scale, so with great metabolic costs. Similar periodic destruction and regeneration of the epithelium in the female reproductive system is well known across the animal kingdom, including human beings and in the primates during menstruation, and in the lower mammals during estrus 47 . It seems probable that in chernetids, the cells remnants of degenerating epithelial cells are not wasted but become transferred to the brood sac in the second round of the nutrients provisioning. It needs to be confirmed in future studies.
Another interesting issue worth considering is the mode of nutritive fluid secretion. The epithelial cells responsible for this process accumulate in their cytoplasm a huge number of secretory granules that exhibit a heterogeneous structure. The process of secretion is quite complex and generally well fits the apocrine mode (see e.g., 48,49 ). Secretion starts with defragmentation of the apical part of the cell, proceeds by releasing secretory granules into the lumen of respective organs (postovulatory stalks, ovaries, and oviducts), and eventually ends with the appearance of the homogeneous nutritive fluid. During this process, the height of the epithelial cells gradually and significantly decreased. It remains puzzling what is the mechanism of the transformation of the secretory material from the granular to the fluid form. Future studies are required to answer the questions of whether it is the result of the secretion of enzymes that extracellularly "digest" the released material, or whether secretory granules "dilute" their content due to a liquid transferred from the hemolymph to the lumina of the ovaries and oviducts.

Conclusions and future perspectives
The results of the study confirm our research hypothesis that in chernetids the secretory phase of the ovarian cycle strongly resembles that described in Chelifer cancroides. We clearly showed that chernetids, like C. cancroides, are characterized by efficient provisioning the embryos with nutrients produced by three populations of hypertrophic and polyploid epithelial cells, that is the postovulatory stalk cells, the epithelial cell of the ovary and oviduct wall. Our investigations also provide a complete step-by-step description of spectacular alterations that occur in the ovary and oviduct during two consecutive stages of the ovarian cycle, including a new set of data: i. postovulatory stalks are the most voluminous structures in the ovary and they reach their maximum size when become filled with the nutritive fluid; ii. secretion of nutrients starts with releasing heterogenous secretory granules, and finishes with diminishing the height of hypertrophic and polyploid cells and transformation of the secretory granules into the homogenous liquid; iii. polyploid epithelial cells of the ovarian and oviductal wall undergo a massive degeneration after completion of their secretory activity; iv. epithelium renewal in the ovary and the oviduct occurs due to mitotic activity of the somatic cells that have remained unchanged by hypertrophy and polyploidization.
Although our knowledge of matrotrophy in pseudoscorpions is gradually increasing there are still many gaps and unanswered questions. One of the fundamental questions is how has matrotrophy evolved in this taxon? To address this question, future comparative studies are required to show how the adaptations for matrotrophy look in basal families and the families distantly related to Cheliferidae and Chernetidae.  (Hermann, 1804). The specimens of A. wideri were collected in spring 2019 in SW Poland, cultured at room temperature and fed with the larvae of a firebrat, Thermobia domestica (Packard, 1873) (Insecta: Thysanura: Lepismatidae). The specimens of C. hahnii, Lamprochernes sp., P. scorpioides were collected in summer 2020 in Bratislava, Slovakia. The specimens of P. lacertosus came from terrarium in Bratislava and were collected in spring 2021.

Material and methods
The vouchers are deposited in the University of Wrocław (Department of Animal Developmental Biology).

Light and transmission electron microscopy.
Whole mount observations of the reproductive system were conducted using an Olympus SZ61 microscope equipped with SC30 camera and Stream Start 1.6.1 software. For histological and ultrastructural observations, the ovaries were dissected and fixed in 2.5% glutaraldehyde in 0.1-M phosphate buffer (pH 7.4) for at least 24 h or longer periods (usually for a few days) at 4 °C. For more details see 27,32 . Histochemical analyses. Detection of microfilaments, DNA, and plasma membranes. For detection of microfilaments, DNA and plasma membranes the ovaries were fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 1 h and rinsed in PBS. For detection of microfilaments, the material was stained with 2 mg/ ml Alexa Fluor 488 Phalloidin (Invitrogen, A12379). For DNA detection, the material was stained with DAPI (4ʹ,6diamidino-2phenylindole dihydrochloride). For detection of plasma membranes, the ovaries were stained with Wheat Germ Agglutinin Texas Red X Conjugate (Invitrogen, W21405). For more details see 27 .
Detection of endoplasmic reticulum. For detection of endoplasmic reticulum, ovaries were dissected in phosphate buffered saline (PBS) and stained with ER-Tracker™ Red (BODIPY™ TR Glibenclamide) (Invitrogen E34250) according to the manufacturer's protocol. After staining, ovaries were rinsed in PBS and wholemounted onto microscope slides and examined with an Olympus FluoView FV1000 confocal microscope (RRID:SCR_016840). Images were analysed using GIMP (GNU Image Manipulation Program, RRID:SCR_003182). Figures were prepared in Inkscape (RRID:SCR_014479).

Data availability
Data are available on request from the authors.